Micro‐diffusely abnormal white matter: An early multiple sclerosis lesion phase with intensified myelin blistering

Abstract Objective Multiple sclerosis (MS) is a chronic central nervous system disease whose white matter lesion origin remains debated. Recently, we reported subtle changes in the MS normal appearing white matter (NAWM), presenting with an increase in myelin blisters and myelin protein citrullination, which may recapitulate some of the prodromal degenerative processes involved in MS pathogenesis. Here, to clarify the relevance of these changes for subsequent MS myelin degeneration we explored their prevalence in WM regions characterized by subtly reduced myelination (dubbed as micro‐diffusely abnormal white matter, mDAWM). Methods We used an in‐depth (immuno)histochemistry approach in 27 MS donors with histological presence of mDAWM and 5 controls. An antibody panel against degenerative markers was combined and the presence of myelin/axonal aberrations was analyzed and compared with the NAWM from the same cases/slices/regions. Results mDAWM‐defined areas exhibit ill‐defined borders, no signs of Wallerian degeneration, and they associate with visible veins. Remarkably, such areas present with augmented myelin blister frequency, enhanced prevalence of polar myelin phospholipids, citrullination, and degradation of myelin basic protein (MBP) when compared with the NAWM. Furthermore, enhanced reactivity of microglia/macrophages against citrullinated MBP was also observed in this tissue. Interpretation We report a new histologically defined early phase in MS lesion formation, namely mDAWM, which lacks signs of Wallerian pathology. These results support the prelesional nature of the mDAWM. We conceptualize that evolution to pathologically evident lesions comprises the previously documented imbalance of axo‐myelinic units (myelin blistering) leading to their degeneration and immune system activation by released myelin components.


Introduction
2][3] A prominent pathological feature of this disorder is the loss of axon-enwrapping myelin sheaths both in white (WM) and gray matter, a condition that leads to the formation of the sclerotic plaques. 4These aberrations represent a well-characterized final stage of myelin degeneration.However, little is known about the events occurring in seemingly normal WM (e.g., normal appearing WM, ª 2024 The Authors.Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. NAWM) that culminate in myelin disintegration/lesion formation.Recent investigations showed that, in comparison with controls, the MS NAWM presents with enhanced prevalence of typical myelin blisters where the myelin detaches from its enwrapped axon. 2,5This hampers the functioning of the axo-myelinic synapse. 6This synapse is shown to be key to the support of axonmyelinic functionality enabling a glutamate-mediated coupling between myelin and its underlying axon via myelin Ca 2+ increase. 6This change at the level of myelin blistering in MS is further accompanied by a number of morphologically and biochemically relevant alterations, occurring in the absence of overt inflammation, that involve: (i) nodes of Ranvier/nodal protein aberrations, 5 (ii) altered myelin lipid polarity and carbon-hydrogen shifts, 5,7 and (iii) myelin protein citrullination. 5Nodal alterations might influence axonal conduction, increasing the axonal Ca 2+ entrance, 8 leading to myelin lipid biochemical changes which over-activate the protein arginine deiminases (PADs) enzymes, 9 with consequent myelin basic protein (MBP) citrullination. 10The latter event, previously reported in pre-immune demyelination stages in cuprizone mouse models of MS, 11 can contribute to enhanced myelin debris immunogenicity 12 and myelin lamella decompaction, a phenomenon associated with higher myelin vulnerability to protease attack 13 and myelin-associated glycoprotein hydrolysis-related myelin detachment from axonal processes. 14evertheless, whether this sequence of events is also relevant to early demyelination in MS brains remains uncertain.To address this, we concentrated our analysis on patches of microscopic diffuse abnormality in the MS NAWM, which may represent subtle manifestations of ongoing demyelination.These areas, which we coined microscopically manifest diffusely abnormal white matter (mDAWM), share similarities with the classically imaging/histologically defined DAWM, 15,16 although expanding to smaller surfaces in the NAWM (tenths to tens of mm 2 ).Moreover, they show an association with inner veins, 17 blood vessels around which the myelin abnormality spans as in the most established WM lesions, 18 and they cannot be further explained by overt Wallerian degenerative pathology 15 or ongoing remyelination (in comparison with the frank remyelinating/remyelinated MS shadow lesions 19 ).Interestingly, the mDAWM presents with pathological mechanisms previously reported in the NAWM, 5,7 including a myelin blister increase, higher presence of the myelin polar phospholipids and enhanced MBP citrullination.Altogether, the mDAWM might represent a candidate to further explore the early degenerative events connected to MS WM lesion formation, emphasizing myelin blisters as important early drivers of demyelination.

Donor material
Paraffin-embedded brain blocks encompassing subcortical (Sc), deep white matter (dWM), and periventricular (PV) WM from 27 progressive MS and 5 control donors (Table 1) were provided by the Netherlands Brain Bank.No difference in the mean age at death (MS: 64.48  The average MS case disease duration was 27.32 AE 2.65 years.The tissue inclusion was guided by the histological presence of areas of microscopic diffuse abnormality (Fig. 1).The whole study was performed in strict compliance with ethical requirements of the Amsterdam University medical centers/VU medical centrum, Amsterdam.Informed consent was asked to the donors.

Material preparation
Per case all available brain blocks were sliced in 10-umthick slices, set on glass slides and deparaffinized (3 9 10 min in xylene, followed by ethanol 95%-70%, 5 9 5 min).Antibody details for immunohistochemistry are shown in Table 2.

Fluorophore immunohistochemistry
To stain the tissue using fluorophores, during the second day of staining either a direct incubation with secondary fluorescent antibodies (Alexa 488 or 594, 1:400, Thermo-Fisher, US) or an incubation with biotinylated secondary antibodies followed by ABC/Envision kit (Agilent, US) and tyramide precipitation or streptavidin+tyramide protocols (in presence of 0.0015% H 2 O 2 , Tyramide Alexa 488 or 594, ThermoFisher, US) was carried out.All the slices underwent DAPI nuclear staining (1:1000, 5 min; Agilent, US) and were covered using mowiol (Sigma Aldrich, US) plus DABCO.To assess signal specificity during fluorophore and DAB experiments negative and positive (e.g., tonsil staining for CD20 + and CD3 + cell analysis) controls were added when appropriate.

Image acquisition
Whole slides were first scanned (Vectra Polaris, Perkin Elmer inc., US) and digitalized for WM assessment and MHC-II + cell quantification.Same procedure was used

Image analysis
To perform our analysis we selected the regions of interest (ROIs) from slide-scanned images using Qu-Path software 22 and post-process the whole images for quantification using Fiji Image-J (NIH, US).

mDAWM quantification
mDAWM selection was performed screening LFB-and PLP-stained slices (from MS and controls) under the microscope and digitally, searching for areas of ill-defined borders of lower myelination in comparison with the surrounding NAWM.We excluded regions in the close proximity to lesions (<1 mm), guiding our selection using structural postmortem magnetic resonance imaging (MRI) performed before block excision. 23MRI inspection did not reveal the presence of classically defined-DAWM in the region of excision or in the surroundings. 15Moreover, to reduce the artifact risk we considered as mDAWM only those areas whose surface was higher than 0.1 mm 2 and excluding regions whose myelin density change could be otherwise defined by variation in fiber orientation.Myelin density quantification was performed adapting a previously used protocol, 20 which involved ROI selection and manual segmentation to retain the maximum amount of pixel associated to myelin signal.
To confirm the quality of the mDAWM selection, random digitally acquired ROIs of the tissue were automatically processed using the Image-J algorithm percentile (Fig. 1) and the majority of the sections was further cross-sectional inspected to estimate the mDAWM extension in tissue-depth (data not shown).

Vein analysis in mDAWM
Vein + mDAWM areas were visually classified looking for either an orthogonal or parallel inner vessel from which the mDAWM expands in at least two directions.Further inspections of the form of the vessel (e.g., tunica media thickness) were executed to exclude arteries/arterioles from analysis.

Wallerian degeneration analysis
Estimation of Wallerian pathology in mDAWM tissue was carried out acquiring a maximum of four random images (xy = 200*150 lm) from NAWM and mDAWM and analyzing them using Fiji Image-J algorithms, surface analysis plug-ins (percentage of area stained by antibodies against SMI312), and count plug-ins (amount of axonal blebs-blebs/lm 2 ).

Myelin blister quantification
To quantify the blister-, bleb-and axonal degenerative swelling-prevalence we used a previously published protocol 5 (with some adaptations), by which four random confocal images (xyz: 153.9*153.9*0.1 lm) were acquired and myelinated axons stratified according to predetermined criteria. 5Thereafter a random/blind selection of visible MHC-II + cells per image was followed by background subtraction and automatic segmentation.A skeletonize protocol and a skeleton script were run to stratify the sample according to its shape using previously reported criteria. 26Upon stratification of our sample in ramified (long processes, small cell body); hyper-ramified (short processes, wide cell body); rod-like (bipolar processes, large cell body), and amoeboid (macrophage-like shape) cells we analyzed the presence of citrullinated MBP in the different categories (+ vs. À) and evaluated the degree of positivity analyzing the percentage of citrullinated MBP in the different cells.

Image presentation
For representative purposes some DAB stained images underwent color deconvolution using standard image-J plug-ins and representative fluorescent/confocal images have been cropped and brightness/contrast adjusted without altering the region comparability.

Exclusion criteria
Slides whose staining was unsatisfactory were either restained or excluded from the study.Two cases from the original selection were excluded from final analysis due to the lack of satisfying clinical/histopathological assessment.

Statistics
Parametric/nonparametric t-tests (preceded by Shapiro-Wilk or Kolmogorov-Smirnov test), one-way ANOVA or chi-squared/Fisher's tests were used, when appropriate.In case of not-homogeneous variance a ratio t-test was used.ANOVA's were preceded by Spearman's and Bartlett's test for homoscedasticity and followed by multiple comparison post hoc correction.Correlation analysis was performed using Spearman's test.Data were analyzed using Prism 9.0 (GraphPad software, US) and considered significant when the p-value was lower than 0.05.

MS mDAWM shows limited expansion and signs of weak inflammation
In search of candidate regions to study early degeneration stages of MS WM we concentrated on microscopic regions of subtle myelin density reduction in the NAWM, and which we therefore dubbed as mDAWM.To assess the mDAWM we investigated LBF-and PLP-stained brain tissue in a cohort of progressive MS cases (primary progressive n = 3; secondary progressive n = 15; average duration of disease 26.22 AE 3.33 years, Fig. 1A).In total we used 30 blocks that came from frontal (60%), temporal (30%), and parietal (10%) regions of 18 cases.Further exploration of material acquired from five controls showed no frank signs of mDAWM in non-MS tissue.
The mDAWM typically presents with ill-defined borders, variable LFB-staining reduction, and small surface area (Fig. 1A,B; mean extension ROIs = 2.65 AE 0.29 mm 2 ; range ROIs = 0.1-13.89mm 2 ; cutoff 0.1 mm 2 ).Overall, myelin density analysis in the mDAWM reports a significantly reduced myelin staining (LFB) when compared with the matched NAWM and control WM (Fig. 1C).Furthermore, the analyzed mDAWM contains weak increase in MHC-II reactivity (Fig. 1D,E) which negatively correlates with myelin density in the same region and no increase in MHC-II + clusters.These results are indicative of an augmented microglia/macrophage presence, as the count of CD20 + B-lymphocytes did not differ between NAWM and mDAWM (Fig. 1F-J).Furthermore, no increase in the density of CD3 + T-lymphocytes was found in the mDAWM when compared with NAWM (Fig. 1K,L).

mDAWM in MS lacks remyelination, WD, and preferentially extends around inner veins
To clarify whether the selected mDAWM was not the result of ongoing remyelination or Wallerian degeneration ª 2024 The Authors.Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.

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we studied the lesion morphology and the presence of newly myelinating oligodendrocytes (using the marker BCAS-1).First, histologic appearance of the mDAWM showed substantial shape and boundary differences compared to MS shadow lesions (Fig. 2A).Second, the BCAS-1 + cell density was similar between mDAWM and control tissues (NAWM and control WM), and significantly lower than remyelinating shadow lesions (Fig. 2B,C).Moreover, as shown in Figure 2D,E neither axonal density nor the estimation of axonal swellings (blebs) differed between mDAWM and NAWM, suggesting that the mDAWM is less likely the result of Wallerian pathology.Finally, the mDAWM showed a clear association (59% of total mDAWM analyzed, no difference between Sc-, dWM, and PV WM) with the presence of inner veins (Fig. 2F-H), which share similarities with the well-reported central vein present in MS WM lesions, 17,18 and which suggest the possible link between mDAWM and temporally evolving areas of frank demyelination.

Increased prevalence of myelin blisters in the mDAWM
To investigate whether the subtle myelin loss in mDAWM is associated with an exacerbation of the morphological alterations previously observed in the MS NAWM, 5 we quantified the prevalence of myelin blisters in this area.Figure 3A,B depicts the presence of typical myelin swellings in the mDAWM.In line with our NAWM observations 5 the mDAWM also hosts recognizable blisters, blebs, and axonal degenerative swellings (Fig. 3C,D).In total 859 myelin swellings were annotated and analyzed (NAWM: 412; mDAWM: 447).In accordance with our expectations, the mDAWM is characterized by 20% higher prevalence of blisters than in matched NAWM.This increase was not reported when quantifying axonal blebs and axonal degenerative processes (Fig. 3E,F) supporting myelin blister relevance in very early stages of reduced WM myelination.

Phospholipid and MBP posttranslational modifications in the mDAWM
Potential triggers for blister forming in MS NAWM are (i) prodromal phospholipid polarity alterations and (ii) augmented posttranslational modification at the level of MBP (e.g., citrullination). 5,7Due to the increased blister prevalence in the mDAWM we expected that these mechanisms are also more apparent in this region when compared with the NAWM.To clarify these aspects we first stained our material with antibodies against lyso-phosphatidylcholine, a highly polar membrane phospholipid whose conversion from phosphatidylcholine is instigated by a Ca 2+ -mediated translocation of cytoplasmic phospholipase 2. Lysophosphatidylcholine was found to induce demyelination when externally infused in mouse brains 27 and it can alter membrane curvature, making it a candidate for myelin blister formation. 28Analysis of the overall expression of lyso-phosphatidylcholine revealed its augmented presence in MS mDAWM versus the NAWM (Fig. 4A,B) where it is also associated with clear myelin swellings (arrow in inset Fig. 4A).Besides, a documented effect of lysophosphatidylcholine conversion is also the higher activation of the protein arginine deiminase enzyme PAD2, which is responsible of myelin protein citrullination. 10Accordingly, analysis of our samples following citrullinated MBP staining showed an increase in the presence of this posttranslationally modified form of MBP in the mDAWM versus the NAWM (Fig. 4C,D).This effect is accompanied by a similar, albeit more modest, increase in degraded forms of MBP (reminiscent of myelin debris) in the mDAWM when compared with the NAWM (Fig. 4E,F).These findings hint at an exacerbation of MS-relevant chemical changes at the level of myelin proteins prior to myelin fragmentation in the mDAWM.Aiming at a sharper look at myelin protein citrullination in prodromal phases of myelin pathology in MS mDAWM we further performed an analysis on single myelinated axons (Fig. 5A) in the mDAWM.As shown in Figure 5B the proportion of citrullinated/normal MBP is significantly altered in the mDAWM, suggesting that a greater surface of myelinated axons hosts such modified form of MBP in comparison to axons in the NAWM.Intriguingly, the same analysis at the level of comparable axons in the shadow lesions showed an inversion of the citrullinated MBP presence in this area, endorsing a putative specific role of MBP citrullination in the early degenerative/prelesional stages in MS.Similar analysis did not show a clear increase in degraded MBP at axonal level, arguing that the previously observed increase in degraded MBP in the mDAWM might be indeed attributed to debris of degenerated myelin constituents (Fig. 5B).Interestingly, while previous studies from our group did not highlight specific associations between enhanced NAWM citrullination and blister/swelling formation in MS 5 (confirmed here), this effect was clearly prevalent in the mDAWM (Fig. 5C,D), hinting at an intimate relationship between the decompaction of myelin sheaths, presumably caused by the sustained citrullination of MBP, and the increased blistering in the mDAWM.Finally, to test whether the enhanced presence of citrullinated MBP in the mDAWM can form a possible trigger of reactive microglia states against myelin constituents we investigated the topical association between MHC-II + microglia (although we cannot exclude that this marker labels monocyte-derived macrophages) with this altered form of MBP in NAWM and mDAWM.A sample of 286 MHC-II + cells (NAWM: 148; mDAWM: 138) was randomly selected, and further stratified using skeleton scripts in ramified (slightly active) and active (Fig. 5F).As expected, our selected samples contained more ramified cells in the NAWM (18.9%; 28 out of 148) than the mDAWM (2.2%; 3 out of 138).The overall amount of active microglia was further classified into hyper-ramified (NAWM: 25%; 37 out of 148; mDAWM: 34.8%; 48out of 138), amoeboid (NAWM: 14.2%; 21 out of 148; mDAWM: 33.3%; 46 out of 138), and rod-like cells (NAWM: 41.9%; 62 out of 148; mDAWM: 29.7%; 41 out of 138, Fig. 5F).Overall, the mDAWM contains more citrullinated MBP + cells than the NAWM (NAWM: 40%; mDAWM: 57%).Analysis stratifying the sample to include only active MHC-II + cells confirmed this increase in the mDAWM (NAWM: 37%; mDAWM: 58%, Fig. 5G) where a significant difference was particularly spotted when comparing the hyper-ramified (NAWM: 41%; mDAWM: 65%) and rod-like cells (NAWM: 37%; mDAWM: 59%, Fig. 5G).Interestingly, when stratifying our sample based on the level of positivity to citrullinated MBP (from weakly positive to extremely positive), only rod-like MHC + cells in the mDAWM had a different  distribution than in the NAWM (Fig. 5H), being associated with higher content of this modified form of myelin proteins.Overall, this result support an active microglia engagement in the process of highly immunogenic myelin debris phagocytosis in the mDAWM.

Discussion
The work in this publication builds on the inside-out concept, which states that MS starts in the CNS and that autoimmunity is the consequence of the (excessive) release of (posttranslationally modified) myelin fragments. 1,2Earlier work revealed presence of myelin blisters in MS brain areas devoid of immune-related aberrations. 5In this study we go further back on the pathology timescale to gain insight into factors contributing to myelin blistering.
Although studies employing imaging, 29 microscopy, 5,30-33 and biochemistry techniques, 5,7,34 have highlighted the abnormal nature of the NAWM in MS brains, the relevance of such aberrations for the formation of overt lesions remains unknown. 1 Here we describe the presence of axomyelinic unit-relevant destabilization features outside the NAWM limits, where small regions of diffuse abnormality (mDAWM), characterized by modest tissue inflammation 35 , host an exacerbation of such events.Compared with the NAWM, the mDAWM shows (i) myelin blister increase, (ii) enhanced expression of polar phospholipids, (iii) increased presence of citrullinated/degraded MBP, and (iv) heightened phagocytosis of posttranslationally modified myelin constituents.These findings, together with the absence of Wallerian degeneration, a hallmark of the classically defined DAWM, 15,16 and the association with a vein located in the innermost part of the area, a feature similar to that of the central vein in WM lesion formation, 17,18 make of the mDAWM a likely separate (very) early pathological aspect of MS brains able to recapitulate the pathophysiological cascade of events leading toward more frank demyelination stages.This latter concept aligns with recent investigations into the DAWM of progressive MS patients 36,37 and, more importantly, with the abovementioned inside-out hypothesis of MS origin. 1,2,5lthough high-throughput live-cell imaging studies are needed to confirm this sequence of events, we interpret the correlation between high MHC-II + scores and myelin reduction in the mDAWM as supportive of the inside-out paradigm of MS origin.In fact, in our specimens only the highest score (Score 3) seems to clearly associate with more severe myelination drops, an effect accompanied by the lack of significant difference between the amount of MHC-II + cells in the NAWM versus the mDAWM.This finding, combined with the lack of a specific increase of both B and T lymphocytes in the mDAWM when compared with NAWM, contradicts the hypothesis that a massive primary recruitment of inflammatory cells causes tissue demyelination, proposing that heightened microglia reactivity secondarily intervenes to clear up myelin debris when demyelination processes have already started.Supportive of this notion is our result on citrullinated MBP microglia/macrophage phagocytic activity (see below), which might precede the formation of the well-reported nodules of microglia. 24In line with this possibility we did not retrieve a substantial increase of such nodules in mDAWM when compared with NAWM.Interestingly, this feature is in line with studies on other (neuro)degenerative diseases, like atherosclerosis and Alzheimer's disease, which are characterized by primary tissue damage and secondary inflammatory response operated by immune cells. 38,39ue to the low emergence of new lesions in advanced secondary progressive MS cases, 4,40 41 and by studies on living secondary progressive subjects that highlighted a clear evolution overtime from DAWM to focal WM lesions 37 which correlates with the clinical progression of the disease. 36ur myelin blister results further support the prelesional/early lesional nature of mDAWM.Myelin blisters are emerging as important MS pathology correlates, being spotted in several NAWM and peri-lesion areas of MS brains 5 and, more recently, in the optic nerve, 33 a region often associated with the earliest MS lesion formation. 42onetheless, in line with the early involvement of myelin blisters in lesion formation, studies employing cuprizone mouse models of MS have reported the presence of similar morphological alterations in mice weeks prior to demyelination. 30lthough the precise mechanistic dissection of blister formation in MS brains and their relevance for the mDAWM origin are still under investigation, we speculate that early destabilizing events at the level of axon-myelinic unit may involve altered nodes of Ranvier elongation and Na + abnormalities, 5 substantiated by augmented Na + concentrations in both NAWM 43 and DAWM 44 of MS patients.These aberrations may trigger an uncontrolled myelinic Ca 2+ entrance 6 which exacerbates the pathological biochemical myelin sheath changes.Notably, augmented Ca 2+ might activate the formation of lysophosphatidylcholine, a highly polar phospholipid, 45 which, in turn, reduces the amount of Ca 2+ needed to stimulate PAD-2 activity, 9 which catalyzes myelin protein citrullination. 46Interestingly, studies reported the ability of lyso-phosphatidylcholine infusions in mice to promote myelin blistering 27 and the presence of prodromal lipid polarity abnormalities in MS NAWM. 7In accordance with these findings, we observed an enhanced lysophosphatidylcholine and citrullinated MBP reactivity in mDAWM tissue when compared with the NAWM of MS brains, a concept that corroborates the early myelin biochemical change exacerbation in the evolution toward overt states of lesion.Intriguingly, myelin protein citrullination, largely present in MS brains and meninges 5,47 is shown to precede demyelination and autoimmune reaction in cuprizone mouse model experiments, 11 drive the progression of CNS pathology in experimental autoimmune encephalitis in marmoset monkeys, 48 trigger the detachment of myelin lamellae, 49 and contribute to render the myelin debris more immunogenic. 12lthough the enlargement of lamella spacing may promote states of intra-myelinic oedema, 50 representing a confounding factor for our study, we tend to disregard this circumstance in our mDAWM dataset.In fact, our observations do not align with the tissue sponginess previously reported in oedemas, 50 and the expansion and form of the retrieved blisters/swellings share remarkable similarities with those previously found in the NAWM. 5 On the other hand, the citrullinated MBP-dependent sheath detachment potentially makes the myelin more vulnerable to protease  1) Altered Na + homeostasis produces an aberrant Na + influx in dysfunctional nodes of Ranvier in MS.Consequently, the Na + /Ca 2+ exchanger might be pushed to work in a reverse fashion 8 leading to increase of axonal Ca 2+ and higher release of glutamate to the myelin.(2) Over-stimulation of NMDArs produces Ca 2+ overload in the myelin, instigating the translocation of cPLA 2 with conversion of lyso-PC from PC. (3) Lyso-PC reduces the Ca 2+ necessary to activated the PAD2 with consequent citrullination of MBP and activation of the calpain-cathepsin axis (4).Cathepsin-L degrades the MAG promoting the formation of myelin blisters.(B) In the mDAWM an exacerbation of the cascade of mechanisms instigates progression of deterioration with consequent heightened prevalence of blistering of the AMS and higher citrullination of the MBP and myelin debris (dMBP).More citMBP and dMBP are released as myelin constituent debris (5) against whom rod-like microglia ( 6) is particularly involved in engulfing the PTM proteins.(C) Schematic overview of the sequence of degenerative processes moving from the WM of healthy controls (as previously reported using F95 a-peptidylcitrulline antibodies 5 ) to MS NAWM, mDAWM, peri-lesional WM and remyelinating SLs.Citrullination of myelin proteins follows a gradual increase until lesions are formed to reduce its presence in case of remyelination.At the same time myelin blister prevalence increases when MS WM lesions are forming.citMBP, citrullinated MBP; cPLA2, cytoplasmic phospholipase A2; dMBP, degraded MBP; Glu, glutamate; HIP, hyper-ramified microglia; MAG, myelin associated glycoprotein; Nav1.6, voltage-gated sodium channel 1.6; NMDAr, n-methyl-d-aspartate receptor; PAD-2, protein arginine deiminase-2; RAM, ramified microglia.985 attack, 51 an aspect involved in degraded myelin-associated glycoprotein formation 14 and, allegedly, blister formation. 5herefore, it is tempting to speculate that via an increased myelin citrullination in the mDAWM, substantiated by the here reported stronger association between citrullinated MBP and swelling formation in the same tissue than in the NAWM, more blistering and more myelin degeneration is deciding the fate toward WM lesion in MS brains.In response to this a secondary immune reaction might intervene, strongly triggered by citrullinated myelin constituents (Fig. 6A,B).In line with this, heightened microglia/macrophage activity in the mDAWM presents with phagocytic activity against citrullinated MBP.Interestingly, after morphology-based stratification, we found a particular reactivity of rod-like-shaped microglia cells in the mDAWM against citrullinated myelin.Although the function of these specific cell types is still misterious, 52 recent studies have underlined their role in the transition from ramified forms of microglia to an amoeboid macrophagelike activation state, 53 together with their phagocytic features, 54 and their scarce involvement in proinflammatory cytokine production. 55Therefore, a parsimonious interpretation of our results might involve a prompt conversion of inactive state microglia into actively phagocytic phenotypes in response to the increase in highly citrullinated production of myelin debris in the mDAWM.This interpretation aligns with studies showing rapid in vitro transition of this microglia type into amoeboid states when exposed to lipopolysaccharide, 55 although it cannot exclude the property of rod-like-shaped microglia to shift back to a ramified/less active state. 56,57o conclude, in this study we highlight an exacerbation of the pathological hallmarks which we previously spotted in the NAWM of MS brains, 5 showing that, via an (i) increase of posttranslational myelin modifications (which are likely specific for prelesional stages of MS WM, Fig. 6C), (ii) myelin blister enhancement and (iii) microglia-directed response toward citrullinated MBP, the NAWM might encounter a degenerative evolution toward microscopic diffusivity, here dubbed as mDAWM.This evidence represents a valuable aspect to dissect the cascade of prelesional events that leads to the formation of focal lesions in MS brains, and to characterized early degenerative WM areas to intervene early in progressive forms of MS, for which limited pharmacological approaches are available.

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Figure 6 .
Figure 6.Mechanistic model.(A) Schematic depiction of the cascade of pathological events involved in myelin blister formation in the NAWM, as proposed in Luchicchi et al. 5 with some adaptations.(1) Altered Na + homeostasis produces an aberrant Na + influx in dysfunctional nodes of Ranvier in MS.Consequently, the Na + /Ca 2+ exchanger might be pushed to work in a reverse fashion 8 leading to increase of axonal Ca 2+ and higher release of glutamate to the myelin.(2) Over-stimulation of NMDArs produces Ca 2+ overload in the myelin, instigating the translocation of cPLA 2 with conversion of lyso-PC from PC. (3) Lyso-PC reduces the Ca 2+ necessary to activated the PAD2 with consequent citrullination of MBP and activation of the calpain-cathepsin axis (4).Cathepsin-L degrades the MAG promoting the formation of myelin blisters.(B) In the mDAWM an exacerbation of the cascade of mechanisms instigates progression of deterioration with consequent heightened prevalence of blistering of the AMS and higher citrullination of the MBP and myelin debris (dMBP).More citMBP and dMBP are released as myelin constituent debris (5) against whom rod-like microglia (6) is particularly involved in engulfing the PTM proteins.(C) Schematic overview of the sequence of degenerative processes moving from the WM of healthy controls (as previously reported using F95 a-peptidylcitrulline antibodies 5 ) to MS NAWM, mDAWM, peri-lesional WM and remyelinating SLs.Citrullination of myelin proteins follows a gradual increase until lesions are formed to reduce its presence in case of remyelination.At the same time myelin blister prevalence increases when MS WM lesions are forming.citMBP, citrullinated MBP; cPLA2, cytoplasmic phospholipase A2; dMBP, degraded MBP; Glu, glutamate; HIP, hyper-ramified microglia; MAG, myelin associated glycoprotein; Nav1.6, voltage-gated sodium channel 1.6; NMDAr, n-methyl-d-aspartate receptor; PAD-2, protein arginine deiminase-2; RAM, ramified microglia.

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2024 The Authors.Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.

Table 1 .
Summary of the cases used for the whole study.
ª 2024 The Authors.Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.

Table 2 .
List of antibodies, dilutions, and suppliers.Quantification of lyso-phosphatidylcholine, citrullinated/ degraded MBP was obtained either acquiring a maximum of four images per case/region (xy: 200*150 lm, for lysophosphatidylcholine) or equidimensional slide-scanned images (xy: 1.25*0.96mm, for citMBP and dMBP).Single axon analysis on the tissue stained with antibodies against MBP/citrullinated/degraded MBP was performed segmenting the images (xy: 200*150 lm) using a constant auto-threshold algorithm, drawing ROIs around the margin of the analyzed myelinated axons and superimposing the channel associated to citrullinated/degraded MBP staining.Selected myelinated axons were later inspected for swelling presence and stratified, accordingly.
ª 2024 The Authors.Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.LPC, cit/dMBP quantification citMBP content in MHC-II + cells To analyze the content of citrullinated MBP in MHC-II + cells a maximum of 4 confocal images (xyz: 387.88*387.88*0.1 lm) per case/area were acquired.
the late disease stage/ age of our cohort may represent an argument against the association between mDAWM and WM lesion formation.Although more investigations are warranted, our conclusions are in line with extensive histopathological examinations by Luchetti et al. which observed sustained inflammatory and demyelinating activity several decades after the diagnosis in late stage progressive MS cases,