Activated microglia mediate axoglial disruption that contributes to axonal injury in multiple sclerosis

Abstract

The complex manifestations of chronic multiple sclerosis (MS)are due in part to widespread axonal abnormalities that affect lesional and nonlesional areas in the central nervous system. We describe an association between microglial activation and axon/oligodendrocyte pathology at nodal and paranodal domains in normal-appearing white matter (NAWM) of MS cases and in experimental autoimmune encephalomyelitis (EAE). The extent of paranodal axoglial (neurofascin-155(+)/Caspr1(+)) disruption correlated with local microglial inflammation and axonal injury (expression of nonphosphorylated neurofilaments) in MS NAWM. These changes were independent of demyelinating lesions and did not correlate with the density of infiltrating lymphocytes. Similar axoglial alterations were seen in the subcortical white matter of Parkinson disease cases and in preclinical EAE, at a time point when there is microglial activation before the infiltration of immune cells. Disruption of the axoglial unit in adjuvant-immunized animals was reversible and coincided with the resolution of microglial inflammation; paranodal damage and microglial inflammation persisted in chronic EAE. Axoglial integrity could be preserved by the administration of minocycline, which inhibited microglial activation, in actively immunized animals. These data indicate that, in MS NAWM, permanent disruption to axoglial domains in an environment of microglial inflammation is an early indicator of axonal injury that likely affects nerve conduction and may contribute to physiologic dysfunction.

Figure 1

MS normal appearing white matter is characterised by microgliosis, axon damage and perivascular lymphocytic infiltrates. (A, B) Luxol fast blue histology and MOG immunochemistry of forebrain post-mortem tissue blocks was used to identify normal appearing white matter regions of interest (arrow head in A). (C) HLA-DR⁺ microglia with an activated morphology were found throughout the normal white matter and CD3⁺ T-cell infiltrates were restricted to perivascular cuffs (D). (E-E2) Immunostaining with the SMI32 antibody identifies stressed/ damaged axons containing nonphosphorylated neurofilaments morphologically consistent with transections (asterisks) or closely apposed to microglia (IBA1⁺, arrows). Microglial activation was confirmed by their altered morphology and expression of HLA-DR and iNOS in MS normal white matter (F,F1) and were present at a greater density in comparison to controls (G,H). Ctrl; control, MOG; myelin oligodendrocyte glycoprotein, LFB; luxol fast blue. Scale bars, A= 0.5cm; B, C, E-E2= 20μm; D= 100μm; F-F1= 10μm; G, H= 50μm.

Figure 2

Paranodal axo-glial disruption in MS normal appearing white matter. (A) Paranodal axo-glial junctions identified by immunostaining for Nfasc155 normally revealed compact paranodal structures separated by Na_v1 channels of the node, although elongated, disrupted structures were noted (arrows in B). (C, D) Paranodal disruption, which takes the form of an elongation along the length of the axon, was noted by an altered expression in Nfasc155⁺ and Caspr1⁺ immunostaining (arrow in D). (E) Quantification of single Nfasc155⁺ structures from control and MS white matter revealed a significant increase in Nfasc155 paranode length in MS (Box plot of mean paranodal lengths per case showing minimum, maximum, interquartile and median values), ***= p< 0.001, Mann-Whitney test. By comparing Nfasc155⁺ structure length with markers of microglial activation, we found a significant correlation between Nfasc155⁺ structure elongation and HLA-DR⁺ microglial density (F) and iNOS expressing microglia (G); but not to the presence of CD3⁺ T-lymphocytes in the MS tissue (H). Spearman’s non-parametric correlation. Ctrl= control. Scale bars, A-D= 2μm; F-H= 50μm; I-I” = 10μm.

Figure 3

Paranodal disruption is associated with underlying axonal pathology. (A) Nfasc155⁺ paranode length correlated with the incidence of SMI32⁺ axons per NAWM region of interest (Spearman’s non-parametric correlation). (B) Quantification of individual Nfasc155⁺ paranodes revealed an increase in length of those associated with SMI32⁺ axons in comparison to Nfasc155⁺ paranodes on SMI32^- profiles (Box plot of mean paranodal lengths per case showing minimum, maximum, interquartile and median values). ANOVA and Bonferroni’s multiple comparison post test (* p<0.05; *** p<0.001). (C-C2), SMI32⁺ axons associated with disrupted Nfasc155⁺ paranodes and SMI32⁺ axonal swellings were noted at nodes of Ranvier (D). (E) Amyloid precursor protein (APP) accumulation is an indicator of axonal pathology and APP was noted in axons of the NAWM that occasionally associated with disrupted nodal profiles. APP, amyloid precursor protein; NAWM, normal appearing white matter. Scale bars, C-F= 2μm; G= 10μm.

Figure 4

Microgliosis and paranodal disruption results in the displacement of juxtaparanodal domains and the loss of Na_v1.6 expression at nodes. (A-C) K_v1.2 that demarcate the juxtaparanodal domains underneath the myelin sheath are normally separated from the node by paranodes (Nfasc155⁺). Nfasc155⁺ paranodal junctions with a disrupted appearance frequently coincided with an apparent encroachment of juxtaparanodal K_v1 channels (arrows, B, C). MS NAWM was categorised as containing relatively low (MS ‘low’) NAWM inflammation/ axonal pathology or relatively high inflammation/ axonal pathology (MS “high; see methods and Table 1 for details). K_v1⁺ juxtaparanodal profiles were displaced nearer to the node of Ranvier in MS NAWM, with the juxtaparanodal-nodal separation smallest in the MS ‘high’ group (D). Within the same group, the occurrence of overlapping Nfasc155⁺/ K_v1⁺ domains was greatest. Box plot representing mean values/ case showing minimum, maximum, interquartile and median values. ANOVA and Bonferroni’s post test (D, E; * p<0.05; ** p<0.01). (E). Quantification of Na_v1⁺ nodal profiles associated with paranodal Nfasc155⁺ immunolabelling revealed these structures to be unchanged in size between control and MS (F). Na_v1 at the node associate with cytoskeletal proteins such as βIV-spectrin that label nodes of Ranvier (G-I). Immunostaining for the Na_v1.6 channel subtype revealed instances of attenuated or undetectable staining of nodes with normal and disrupted Caspr1⁺ paranodes (J-L). NAWM, normal appearing white matter; JPN, juxtaparanode. Scale bars, A-C= 2μm; G-I,K,L= 1μm, J= 0.5μm.

Figure 5

Microglial inflammation associated with the disruption of paranodal junctions in an animal model of multiple sclerosis. (A,B) Immunohistochemistry for CD11b revealed significant increases in microglial density in transverse sections of spinal cord of MOG_35-55 immunised mice at dpi10 (MOG dpi10) and long term CFA and MOG groups (dpi44). ANOVA with Bonferoni’s post test (n=5 animals per group). (C-E) MOG immunised animals at dpi10 contained meningeal, perivascular and parenchymal TLR4⁺ and iNOS⁺ monocytes/ microglia in lumbar spinal cord. TLR4⁺ microglia were noted in CFA dpi10 tissue but not in the CFA long term animals, indicative of temporary microglial activation in this group. (F) Elongated Nfasc155⁺ paranodes were noted in pre-symptomatic MOG dpi10 tissue (inset) and paranodal elongation was significantly different from naïve in CFA dpi10, MOG dpi10 and long term MOG lumbar spinal cord (G). Nfasc155 changes were reversed in the long-term CFA group. Box plot of mean paranodal lengths per case showing minimum, maximum, interquartile and median values. ANOVA with Bonferoni’s post test (n=5 animals per group). *=p<0.05, **=p<0.01, ***=p<0.001. dpi, days post induction. Scale bars, A= 300μm; C-E= 20μm; F= 2 μm.

Figure 6

Paranodal axo-glial disruption in EAE associates with juxtaparanodal re-arrangements and axon stress. (A) Longitudinal sections of lumbar spinal cord white matter contained K_v1.2⁺ juxtaparanodal and Nfasc155⁺ paranodal domains located in a non-overlapping conformation. MOG immunised mice at 10dpi frequently presented K_v1.2⁺ encroachment into disrupted paranodal domains (B). (C) SMI32⁺ stressed/ damaged axons associated with disrupted Nfasc155⁺ profiles in both CFA and MOG immunised groups at 10dpi. The incidence of overlapping K_v1.2/ Nfasc155 domains were significantly increased in CFA and MOG immunised dpi10 mice in comparison to naïve controls (D; proportion of overlapping K_v1⁺ / Nfasc155⁺ domains of total K_v1⁺/Nfasc155⁺ domains analysed; n=5 per group. ANOVA with Bonferroni post test). (E) Damaged axons (SMI32⁺) associated with significantly elongated Nfasc155⁺ paranodal profiles in comparison to Nfasc155⁺ paranodes upon SMI32-fibres in both CFA and MOG dpi10 groups. (Box plot of mean paranodal lengths per case showing minimum, maximum, interquartile and median values; CFA or MOG group means compared by Mann-Whitney t-test). n=5 animals per group. *=p<0.05, **=p<0.01, ***=p<0.0001. dpi; days post induction. Scale bars A-C= 2μm.

Figure 7

Disruption of nodal Na_v1.6 expression in EAE. (A,B) βIV spectrin⁺ nodes associated with the vast majority of Caspr1⁺ paranodes in MOG immunised dpi10 animals. Nodal abnormalities took the form of Na_v1.6 negative nodes (asterisks) and heminodes (arrows, B) at Nfasc155⁺ paranodes. Na_v1.6⁺ negative nodes were associated with normal appearing and disrupted paranodal profiles (C-E). (F, G) The intensity of Nfasc155 (green line) and Na_v1.6 (red line) immunostaining from regular and Na_v1.6 negative nodes was calculated by plotting the mean of 20 normal and 20 Na_v1.6⁺ negative nodes (pixel intensity, 0-255 units), normalized and averaged from single confocal images. These data confirmed the absence of detectable Na_v1.6 at the node. The proportion of aberrant (Na_v1.6 negative or Na_v1.6 heminodes) nodes were significantly different in MOG and CFA animals at dpi10 (H, I) compared with naïve animals. This trend was reversed in long term CFA animals (H), whilst there was an increased prevalence of aberrant nodes in long term MOG immunised animals compared with naïve controls (I). ANOVA and Bonferroni’s post test (***p<0.001). n=5 animals per group. dpi; days post induction. Scale bars A, B=5μm; C-E=2μm.

Figure 8

Administration of minocycline reduced the density of microglial inflammation and abolished disease induced changes at paranodal and nodal units. (A-C) The density of CD11b immunoreactivity (mean signal intensity per animal± sem) in transverse spinal cord was greatly reduced in minocycline treated mice in comparison to vehicle dosed MOG immunised animals. (D, G) Minocycline treated animals contained microglia with a resting morphology that lacked TLR4 expression, in contrast to microglia in vehicle MOG immunised mice (E, H). (F) Nfasc155⁺ paranode length was increased in vehicle treated animals in comparison to the minocycline group (Box plot of mean paranodal lengths per case showing minimum, maximum, interquartile and median values), whilst the incidence of nodal abnormalities was greater in the vehicle dosed animals (I). n=5 animals per group; *=p<0.05, **=p<0.01 Mann-Whitney test. Dpi; days post induction. Scale bars A, B= 200μm, D-H= 30μm.