Axon-Myelin Unit Blistering as Early Event in MS Normal Appearing White Matter

Abstract

Objective: Multiple sclerosis (MS) is a chronic neuroinflammatory and neurodegenerative disease of unknown etiology. Although the prevalent view regards a CD4⁺ -lymphocyte autoimmune reaction against myelin at the root of the disease, recent studies propose autoimmunity as a secondary reaction to idiopathic brain damage. To gain knowledge about this possibility we investigated the presence of axonal and myelinic morphological alterations, which could implicate imbalance of axon-myelin units as primary event in MS pathogenesis.

Methods: Using high resolution imaging histological brain specimens from patients with MS and non-neurological/non-MS controls, we explored molecular changes underpinning imbalanced interaction between axon and myelin in normal appearing white matter (NAWM), a region characterized by normal myelination and absent inflammatory activity.

Results: In MS brains, we detected blister-like swellings formed by myelin detachment from axons, which were substantially less frequently retrieved in non-neurological/non-MS controls. Swellings in MS NAWM presented altered glutamate receptor expression, myelin associated glycoprotein (MAG) distribution, and lipid biochemical composition of myelin sheaths. Changes in tethering protein expression, widening of nodes of Ranvier and altered distribution of sodium channels in nodal regions of otherwise normally myelinated axons were also present in MS NAWM. Finally, we demonstrate a significant increase, compared with controls, in citrullinated proteins in myelin of MS cases, pointing toward biochemical modifications that may amplify the immunogenicity of MS myelin.

Interpretation: Collectively, the impaired interaction of myelin and axons potentially leads to myelin disintegration. Conceptually, the ensuing release of (post-translationally modified) myelin antigens may elicit a subsequent immune attack in MS. ANN NEUROL 2021;89:711-725.

FIGURE 1

Blistering of AMS presents more frequently in MS than non‐MS WM. (A) Top panel: Example of PLP staining on MS brain sections (brown). The dashed lines indicate a boundary for NAWM, or a WML, or a GML. Bottom panel: Photomicroscope acquisitions of PLP and MHC‐II from the same case show NAWM (*) and a chronic WML (#). Thin‐dotted lines show the lesion borders while thick‐dotted lines mark the periventricular region (PV). (B) Top panel: LFB staining from an MS case. Bottom panel and inset: MHC‐II staining performed on the same case highlights a chronic active lesion (**). (C, D) Examples of PLP and LFB staining images of swelling formations (arrows) retrieved in NAWM (left) and lesion/perilesion (right) of MS cases. (E) Top panel: Cartoons (left), 3D drawings (middle), and examples (right) of the different types of swelling analyzed. Bottom graph: Distribution of swelling types among CTRL, MS, and AD (chi squared test; chi squared ₍₆₎ = 108.6; p < 0.0001). (F) Blister percentage is significantly increased in MS than CTRLs and AD (Blisters: 1‐way ANOVA; F_(3,13) = 12.17; p = 0.0004; Sidak's multiple comparison test; p = 0.031 MS NAWM vs CTRLs; p = 0.0009 MS perilesion vs CTRLs; p = 0.042 MS NAWM vs AD; p = 0.002 MS perilesion vs AD; blebs: Brown‐Forsythe ANOVA; F_(3.0,5.563) = 3.156; p = 0.114 CTRLs vs MS vs AD; degenerative: 1‐way ANOVA; F_(3,13) = 1.675; p = 0.221 CTRLs vs MS vs CTRLs). (G) Top panel: Examples of inflammatory regions in the WM of an encephalitis case. Arrowheads indicate CD3+ cells. Dotted line indicates the ventricular region. Bottom graph: distribution of swelling types between CTRLs, MS NAWM, and encephalitis (ENC) WM (chi squared test; chi squared ₍₆₎ = 108.6; p < 0.0001). (H) Blister percentage is significantly higher in MS NAWM than EN WM (Blisters: 1‐way ANOVA; F _(2,9)=6.672; p = 0.0167; Sidak's multiple comparison test; p = 0.032 MS NAWM vs ENC). Scale bars: 5 mm in A (top panel) and B; 500μm in A (bottom panel); 1mm in B bottom panel (inset scale bar is 200μm); 200μm in C and D (inset is 25μm); 2.5μm in E, 100μm in G. Data in F, H are reported as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. AD = Alzheimer's disease; AMS = axo‐myelinic synapse; ANOVA = analysis of variance; CTRL = control; GM = grey matter; GML = grey matter lesion; LFB = Luxol Fast Blue; MHC II = major histocompatibility complex II; MS = multiple sclerosis; NAWM = normal appearing white matter; PLP = proteolipid protein; WML = white matter lesion.

FIGURE 2

Biochemical and structural fingerprints of MS swellings. (A) Example of CASPR staining to locate the swellings along axonal tract. Graph reports a differential distribution of swelling in our dataset (Nodal‐paranodal/internodal: 83.1%/16.9% and 53%/47% CTRL vs MS; Fisher exact test; p = 0.0001). (B) Graphical representation of CARS analysis (top) and intensity peak graphs of the analyzed regions (bottom). (C) Top graph: Analysis of CH₂ peak in swellings (sw) and peri‐swelling region (pre/post sw) in MS NAWM and CTRLs (CH₂: RM ANOVA; myelin location: F_(1,6) = 8.743; p = 0.025; group effect: F_(1,6) = 6.937; p = 0.039; interaction myelin location x group: F_(1,6) = 2.659; p = 0.154; Sidak's multiple comparison test; t_(12.00) = 3.041; p = 0.020; swelling CTRL vs swelling MS; p = 0.161 pre/post swelling CTRL vs pre/post swelling MS). Bottom graph: Comparison between CH₂ intensity peak in swellings and peri‐swellings versus normally myelinated fibers from controls (Welch's t test; t_(4.713) = 3.485; p = 0.019; normal appearing CTRL fibers vs MS swellings; t_(3.877) = 2.944; p = 0.044; normal appearing CTRL fibers vs MS pre/post swelling). (D) Example of NR2c (top panel) and NR3a (bottom panel) NMDA receptor subunit expression in the swelling formations. (E) Both NR2c and NR3a NMDAR subunits are overexpressed in the MS NAWM swelling formations, compared with controls (CTRL vs MS NR2c CC: Mann–Whitney test; U = 7; p = 0.042; NR3a CC: Mann–Whitney test; U = 7; p = 0.04). (F) Example of MAG staining inside the swellings. (G) While MAG is expressed in MS NAWM swellings (arrow top panel), it is absent in the peri‐swelling region (*). MAG analysis inside the swellings and peri‐swelling regions (bottom panel) reveals a difference between MS and CTRLs (CTRL vs MS NAWM; RM ANOVA: interaction group × location, F_(1,6) = 8.877, p = 0.025; Sidak's multiple comparison test: swelling: p = 0.012; pre/post swelling: p = 0.0002). (H) Left panel: Graphic representation of MAG hydrolysis. Right graph: Analysis showing that in the peri‐swelling region intracellular MAG accumulates in MS (REML mixed effect model: interaction location × group × epitope, F_(1,12) = 13.83, p = 0.003; swelling intra‐ vs extracellular MAG in MS: Tukey's multiple comparison test: p = 0.999; extra vs intracellular MAG pre/post swelling MS: p < 0.0001). Scale bars: 10 μm in A (inset scale is 5 μm), 25 μm in D (inset scale is 10 μm) and 50 μm in F (inset scale is 10μm). In G scale bar is 5μm. Data in C (top graph), E, and G, and H are reported as mean ± SEM, wheras in C bottom graph is mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001. ANOVA = analysis of variance; CARS = coherent anti‐stokes Raman scattering; CASPR = contactin associated protein; CTRL = control; GM = grey matter; GML = grey matter lesion; MAG = myelin associated glycoprotein; MS = multiple sclerosis; NAWM = normal appearing white matter; NMDA = N‐methyl‐D‐aspartate; NMDAR = N‐methyl‐D‐aspartate receptor; PLP = proteolipid protein; REML = restricted maximum likelihood; WML = white matter lesion.

FIGURE 3

(Inter)nodal pathology in MS NAWM. (A) Schematic representation of the main structural/tethering proteins expressed in the AMS. (B) Photomicroscope fluorescent images of Cont‐1 (left) and MAG (right) in the axonal tract. (C) Region of interest (ROI) analysis (expressed as percentage of volume) of cont‐1 and MAG reactivity in CTRLs versus patients with MS CC and sWM, respectively (CTRL vs MS NAWM Cont 1: Welch's t test; t_(17.73) = 5.038; p < 0.0001; MAG: Welch's t test; t_(17.94) = 3.55; p = 0.023) (D) Analysis (CTRL vs MS NAWM) of the expression level of the main tethering proteins across different ROIs of MS (n = 4) and controls (n = 5) reports an increase rather than a decrease of Cont‐1 (sWM: Welch's t test; t_(13.25) = 2.769; p = 0.016), NF155 (sWM: Mann–Whitney test; U = 23; p = 0.043), CASPR (sWM: Welch's t test; t_(10.90) = 5.038; p = 0.0004) Cont 2 (CC: Mann–Whitney test; U = 7; p = 0.0005). (E) Left panel: Example of NF155 (left) and CASPR (right) staining. (F) Graphic representation of the elongated length of the Ranvier's node in MS. (G) Analysis of distance between adjacent NF155 (left graph) and CASPR (right graph) molecules in MS and CTRLs (CTRL vs MS NAWM NF155: Welch's t test; t_(3.610) = 2.970; p = 0.047; CASPR: Welch's t test; t_(6.119) = 4.199; p = 0.005). (H) Examples of Na_v expression in MS NAWM/CTRL WM nodes. From top to bottom: nodal Na_v flanking paranodal CASPR; bilateral Na_v, and unilateral Na_v displacement to the paranode; and 2 cases showing Na_v paranodal invasion patterns with evident nodal Na_v negativity. (I) Analysis of paranodal length in MS and controls (Welch's t test; t_(7.618) = 1,427; p = 0.193; CTRL vs MS NAWM). (J) Distribution of the Na_v/CASPR expression patterns in MS NAWM and control axons (chi squared test; chi squared ₍₃₎=23.04; p < 0.0001; CTRL vs MS). (K) Na_v redistribution in the paranodal region with reduced expression in the nodal region is a specific feature of axons with augmented nodal length (Kruskal‐Wallis test; KW = 24.66; p < 0.0001; Dunn's multiple comparison test; p = 0.023; −+− vs +−+; p = 0.054; +++ vs +−−; p = 0.0003; −+− vs +−−) Scale bars: 5μm in B and 10μm in D. In H are from top to bottom: 5, 10, 5, 10, and 10μm. Data in C and F are reported as mean ± SEM, whereas in J thick dotted lines represent mean and thin dotted lines the SD. *p < 0.05; **p < 0.01; ***p < 0.001. AMS = axo‐myelinic synapse; CASPR = contactin associated protein; CC = corpus callosum; CTRL = control; MAG = myelin associated glycoprotein; MBP = myelin basic protein; MOG = myelin oligodendrocyte glycoprotein; MS = multiple sclerosis; NAWM = normal appearing white matter; PLP = proteolipid protein; ROI = region of interest; sWM = subcortical/periventricular white matter; WML = white matter lesion.

FIGURE 4

Myelin NMDA receptors are dysregulated in MS NAWM. (A) Confocal example of double staining for PLP and NMDAR NR1 obligatory subunit. NR1 is present in myelin (arrows), and cell bodies (DAPI staining *) (B) SMI‐312 (axonal neurofilament), PLP (myelin), and NMDA receptor subunit NR2c triple staining shows preferential distribution of NMDARs in the periaxonal space (arrowhead). Blue color:DAPI staining. (C) Triple staining for NMDAR NR1 and NR3a subunits shows colocalization with the myelin (PLP). (D) NR3a seem mostly expressed in the inner myelin sheath (left panel), whereas NR1 seems ubiquitously expressed in the entire myelin. (E, F) Top panels: Examples of PLP and NR3a subunit co‐expression in the corpus callosum of CTRLs and MS cases. Bottom panels: Magnification of axonal processes reporting co‐expression of PLP, NR1, and NR3a subunits in CTRL (E) and MS cases (F). (G) Analysis of myelin occupied by either NR2c or NR3a in MS NAWM corpus callosum. While expression of NR2c is unaffected (CTRL vs MS NAWM myelin occupied by NR2c: Welch's t test; t_(10.70) = 0.413; p = 0.688; NR2c in myelin: Welch's t test; t_(7.362) = 0.451; p = 0.665) NR3a is overexpressed in MS compared with controls (CTRL vs MS NAWM myelin occupied by NR3a: Mann–Whitney test; U = 4; p = 0.012; NR3a in myelin: Welch's t test; t_(8.060) = 2.577; p = 0.038). Scale bars are 20μm in A; 10μm in B and C and 2.5μm in D. E and F top panel scale bar is 25μm (inset scale bar is 5μm). Bottom panel for E and F scale bar is 10μm Data are reported as mean ± SEM. *p < 0.05. CTRL = control; MS = multiple sclerosis; NAWM = normal appearing white matter; NMDA = N‐methyl‐D‐aspartate; NMDAR = N‐methyl‐D‐aspartate receptor; PLP = proteolipid protein.

FIGURE 5

Citrulline content in MS. (A) Spectral nuance camera examples of citrulline content in CTRL WM and MS NAWM. (B) Citrulline staining co‐localizes with PLP staining (arrowheads). (C) Analysis of the area fraction occupied by citrullinated proteins in MS NAWM versus CTRLs reveals higher content in MS cases (Welch's t test; t_(9.004) = 3.517; p = 0.006; CTRL vs MS NAWM). (D) MS WM blood vessel presents with citrulline content. (E) Meninges in patients with MS also contain citrullinated proteins. Top panel: Image of the leptomeninges of an MS case in a region adjacent to a cortical lesion area. Arrowheads indicate citrullinated proteins accumulating in the meningeal space. Bottom panel: Representative image of a staining performed in a cohort of MS and CTRL cases showing citrulline accumulation (red) in the meninges (arrowheads). (F) Analysis of area (%) occupied by citrulline in the meninges in MS versus controls (Welch's t test; t_(10.77) = 2.496; p = 0.030 CTRL meninges/cortex vs MS meninges/cortex). (G) Example of citrullinated swellings identified by PLP staining. (H) Analysis of citrullinated area in swellings of MS versus controls does not show a significant difference (Mann–Whitney test; U = 7; p = 0.556; CTRL vs MS NAWM). Scale bars are 200μm in A; 10μm in B, 50μm in D, 200μm in E, and 10μm in G. Data are reported as mean ± SEM. *p < 0.05; **p < 0.01. CTRL = control; MS = multiple sclerosis; NAWM = normal appearing white matter; PLP = proteolipid protein; WM = white matter.

FIGURE 6

Proposed sequence of events in MS. Graphic representation of the hypothesized cascade of mechanisms involved in AMS imbalance in MS. Red pathways 1a (as indicated by arrows) shows the possible instigator in AMS imbalance. Elongation of the Ranvier's node might induce a re‐distribution of Na_v channels (eg, the persistent firing channels Na_v1.6 ⁴¹ ). High axonal Na⁺ entrance might promote an NCX ⁴³ inversion increasing the intracellular Ca²⁺ entrance (2, blue pathway), resulting in a higher probability of glutamate release from axon to myelin. At the same time, myelin lipid aberrations (red pathway, 1b) might reduce the axonal shielding from external ions, favoring Ca²⁺ entrance in the axon (where Ca_v are expressed ¹³ ). This effect might also add to that shown in 1a. Higher glutamate release triggers Ca²⁺ increase in the myelin, activating PAD enzymes to citrullinate myelin proteins ¹² (black pathway, 4). Release of citrullinated proteins might instigate an immune response against the debris. Ca²⁺ entrance might also activate the calpain‐cathepsin axis, ³² detrimental for lysosomes, which ultimately would promote dMAG formation ²² (violet pathway, 5). This effect instigates myelin detachment from its axon. Finally, Ca²⁺ increase in the myelin can also favor the translocation of PLA‐Iva enzymes, ²⁹ which affects the lysosomal functionality and lead to lipid biochemical changes. The latter alteration may consequently make the lipid membrane more permeable to external ions (including Ca²⁺, brown pathway, 6). Detachment of myelin causes the retrieved blisters (7), causing AMS instability and hampering the lactate‐dependent signal to axonal mitochondria, ¹³ instigating virtual hypoxia, axonal swelling (8), and, ultimately due to insufficient axonal trophic support, axonal degeneration (9). AMS = axo‐myelinic synapse; dMAG = degraded myelin associated glycoprotein; MAG = myelin associated glycoprotein; MS = multiple sclerosis; PAD = peptidyl arginine deiminase.