Both CD4+ and CD8+ T cells play critical roles in the immunopathogenesis of multiple sclerosis (MS). 1C6 T cell receptor transgenic (TcR-Tg) mice on the nonobese diabetic (NOD) background have a MOG[35-55] (myelin oligodendrocyte glycoprotein 35-55)-specific, major histocompatibility complex class II-restricted TcR that selects for both CD4+ and CD8+ T cells, and we previously reported that adoptive transfer of 1C6 CD4+ T helper 17 (Th17) cells can induce experimental autoimmune encephalomyelitis with a progressive disease course. In the current study, we assessed the function and pathogenicity of 1C6 CD8+ T cells. We found that they proliferated and produced inflammatory cytokines in response to MOG[35-55] peptide under both T cytotoxic 1 (Tc1) and Tc17 differentiation conditions, albeit with reduced expansion relative to their Th1 or Th17 counterparts. Both 1C6 Tc1 and Tc17 cells were able to induce experimental autoimmune encephalomyelitis upon adoptive transfer to NOD.Scid mice. Intriguingly, we noted in vivo expansion of CD4+ T cells in the spleen and CNS of NOD.Scid recipients as well as in lymphocyte-sufficient animals, despite 1C6 Tc cells being purified on CD8 expression prior to transfer. Furthermore, 1C6 Tc17 cells expressed ThPOK, a master differentiation factor for CD4+ T cells. Finally, anti-CD4+ T cell blockade abrogated CD8+ T cell infiltration of the CNS and disease induction in Tc17 recipient mice. Our data provide insight into the interplay of CD4+ and CD8+ T cells in CNS autoimmunity.

Abstract Multiple sclerosis (MS) is an immune-mediated demyelinating disease, with progressive neurodegeneration that is refractory to current therapies. Here, we identify microglia aging as a major factor responsible for age-related loss of beneficial demyelinating and remyelinating brain functions. Brain transcriptomics and in situ spatial gene transcription analysis revealed significant oligodendrocyte loss in both young and aged mice during cuprizone-induced experimental demyelination, but impaired microglial activation in aged mice. Age-related defects in microglial activation were associated with reduced clearance of dead myelin and accumulation of lipid droplets, and impaired remyelination, implying exhaustion of microglial function in the aged mouse brain. Transcriptomic analysis of human brain samples from MS donors with matched disease course and severity and with chronic active and inactive lesions, validated that microglial activation is strongly reduced with increasing age. To investigate microglial responses after repeated demyelinating insults and their direct impact on myelin integrity, we established an experimental model of repeated demyelinating episodes in young and aged mice to recapitulate MS features. Notably, aged mice developed a progressive neuroinflammatory response following sequential demyelinating episodes, in contrast to the alternating cycles of demyelination and remyelination reminiscent of relapsing-remitting MS that were observed in young mice. Microglia depletion and repopulation using a CSF1R antagonist recovered demyelination-remyelination capacity in aged mice. The results indicate that microglia aging is a major determinant in the pathogenesis of progressive MS, and that microglia replacement represents a promising therapeutic approach.

Alois Alzheimer is known for the clinical diagnosis and neuropathological analysis of the neurodegenerative disease named after him. Yet, a less celebrated but equally fundamental contribution lies in his monograph, “Contributions to the knowledge of the pathologic neuroglia and their relationship to degenerative processes in the nervous tissue.” This work, now translated, meticulously details the dynamic role of glial cells in brain pathology, a paradigm-shifting concept for its time. Alzheimer applied then-novel staining techniques -including adaptations of Scharlach red and Mallory hematoxylin- to visualize lipids and protoplasmic degradation in post-mortem human brains. These approaches revealed striking changes in the glial network across disorders ranging from stroke and trauma to multiple sclerosis, syphilis-related dementia, various forms of neurodegeneration, and schizophrenia. He documented the appearance of two novel glial forms, which he termed “ameboid glia” and “glial granule cells”, involved in the phagocytosis of “fatty matter” and “lipoid substances” from degenerating nervous tissue. He also documented “neuronophagia,” where glial cells appeared to directly interact with and “dissolve” damaged neurons. This work laid the conceptual groundwork for modern neuroscience. We now recognize Alzheimer’s “ameboid glia” as the earliest documented observations of activated microglia and astrocytes, and his descriptions of glia’s engagement with cellular damage provided the first evidence for what we now call neuroinflammation. His methodical approach and detailed observations proved that glial cells are not passive bystanders but central, active players that sense, respond to, and shape the course of brain diseases. The English translation and the original German text can be found in the attachment.

BACKGROUND:Aberrant iron homeostasis is increasingly recognized as a key pathological feature in progressive multiple sclerosis (MS). Although the source of excess brain iron remains unclear, haemoglobin is one possible source. To test this hypothesis, we conducted a case-control study to determine whether erythrocytes are more fragile in people with progressive MS (PwPMS) and examined associations between erythrocyte fragility and brain atrophy. METHODS:PwPMS and control individuals were recruited from two centres. Two measures of erythrocyte fragility were assessed at baseline: the Median Corpuscular Fragility (MCF) and haemolysis curve slope. A subset of PwPMS in one centre underwent MR imaging at the same time as osmotic fragility testing and annually for three years thereafter. RESULTS:A total of 174 participants were included (75 PwPMS, 99 controls), with MRI data available for 44 PwPMS. No significant differences in the MCF were observed between PwPMS and controls in either the full or age-matched cohorts. However, the haemolysis curve slope in PwPMS was less steep than healthy controls (median PwPMS = -24.76, controls = -28.73, p = 0.017), consistent with a subpopulation of fragile erythrocytes, with a similar trend in the age-matched subset (p = 0.056). Erythrocyte fragility was associated with normalized whole brain volume at the time of osmotic fragility testing and up to three years thereafter. CONCLUSIONS:Extracellular haemoglobin from lysis of an erythrocyte subpopulation may contribute to neurodegeneration in progressive MS. Further research is warranted to elucidate the interplay between erythrocyte health, inflammation and neurodegeneration, which may open avenues for novel therapeutic strategies.

The complement system is involved in the pathogenesis of inflammatory demyelinating diseases (IDDs) of the CNS. While complement inhibition significantly reduces the relapse rate in neuromyelitis optica spectrum disorders (NMOSDs), no clear consensus has been reached regarding the role of complement in myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) and multiple sclerosis (MS). Therefore, we examined CNS tissues from patients with NMOSD (18 autopsies and one biopsy, median age: 56 years), MOGAD (seven autopsies and 20 biopsies, median age: 34 years) and MS (24 autopsies, median age: 54.5 years) to assess the involvement of the complement system from a histopathological perspective. To investigate complement activity at multiple steps, the tissue deposition of three different complement components (C4d, C3d, and C9neo) was examined using immunohistochemistry. In NMOSD, the typical perivascular rosette/rim pattern of complement deposition was confirmed by the three different complement products within acute astrocyte-lytic lesions. In MOGAD, we observed C4d deposition around perivenous demyelinating lesions in 83% (20/24 tissues). However, C9neo deposition differed between patients, with 73% (11/15 patients with perivenous demyelination-predominant MOGAD) showing limited deposition of C9neo with relatively well-preserved oligodendrocytes (MOGAD type A), while 27% showing strong deposition accompanied by the disappearance of oligodendrocytes (MOGAD type B). The more destructive type B pathology was more frequent among deceased than living patients who, by contrast, had type A pathology in the vast majority. In MS, only C4d showed clear deposits on myelin sheaths in the peri-plaque white matter bordering the edges of the demyelinating lesions. These findings seemed to be characteristic of MS, and the extent and intensity tended to decrease in accordance with lesion activity. Complement deposition in MS lesions was linked to shorter interval between onset and death. These characteristic patterns of complement deposition in the three IDDs likely reflect the distinct pathogeneses of the diseases.

Importance:Differentiating multiple sclerosis (MS) from myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) and neuromyelitis optica spectrum disorder (NMOSD), especially in seronegative cases, remains challenging due to overlapping clinical and imaging features. High-level Epstein-Barr virus (EBV)-derived Epstein-Barr nuclear antigen 1 (EBNA-1) peptide antibody titers may be an MS-specific biomarker that could the improve differential diagnosis. Objective:To determine whether longitudinal EBNA-1 peptide antibodies can distinguish MS from MOGAD and NMOSD. Design, Setting, and Participants:This was a retrospective, multicenter, longitudinal, case-control study with patients from Austria, Germany, and the US. This study assessed samples from 2 independent retrospective cohorts. A test cohort and a validation cohort assessed longitudinal plasma samples from patients with MS, MOGAD, or NMOSD. Patients were recruited between 2001 and 2023 and followed up for 2 years. A combined analysis of both cohorts was conducted in January 2025. Exposures:Plasma EBNA-1 peptide immunoglobulin G (IgG) titers measured by enzyme-linked immunosorbent assay after diagnosis and in 3 follow-up samples. Main Outcomes and Measures:Diagnostic utility of persistent EBNA-1 peptide antibody levels across 4 time points in patients with MS compared with patients with MOGAD and NMOSD. Results:This study included the plasma samples of 2091 patients (mean [SD] age, 31.0 [16.9] years; 1137 female [54.4%]) with neuroinflammatory disease and 1976 healthy controls (mean [SD] age, 39.8 [16.2] years; 1120 male [56.7%]) recruited between 2001 and 2023. The test cohort (310 patients; 54.8% female) included 184 patients with MS, 65 with MOGAD, and 61 with NMOSD (including 12 who were seronegative for aquaporin 4 [AQP4] IgG). The validation cohort (183 patients; 126 female [68.8%]) included 142 patients with MS, 24 with MOGAD, and 17 with NMOSD. In the test cohort, 177 patients with MS (96.2%) had high-level titers in 2 or more of 4 follow-up samples compared with 5 patients (7.7%) with MOGAD (odds ratio [OR], 303.4; 95% CI, 94.4-908.6) and 11 patients (18.0%) with NMOSD (OR, 114.9; 95% CI, 43.0-280.0). Among patients with NMOSD who were seronegative for AQP4-IgG, only 1 (11.1%) had persistent high-level EBNA-1 peptide antibody titers compared with 61 matched patients (96.7%) with MS (OR, 236.0; 95% CI, 18.6-2588.0). In the validation cohort, 135 patients (95.1%) with MS had high-level titers in 2 or more of 4 follow-up samples compared with 4 patients (16.7%) with MOGAD (OR, 96.4; 95% CI, 26.6-293.0) and 3 patients (17.6%) with NMOSD (OR, 90.0; 95% CI, 19.7-319.7). Conclusions and Relevance:Results of this case-control study reveal that persistent high-level EBNA-1 peptide antibody titers may serve as a reliable biomarker for differentiating MS from MOGAD, and NMOSD.

Multiple sclerosis (MS), myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), and neuromyelitis optica spectrum disorder (NMOSD) are immune-mediated inflammatory disorders of the central nervous system (CNS). The temporal relationship between disease-specific autoantibodies and biomarkers of CNS injury before diagnosis remains unclear and is relevant for understanding early pathobiology. Here, we conducted a multicentre retrospective longitudinal case-control study using prediagnostic plasma from 362 individuals who later developed MS, 145 who developed MOGAD, and 60 who developed NMOSD. Plasma IgG levels against CNS antigens, MOG, and AQP4, as well as neurofilament light chain (pNfL), were quantified, and temporal relationships between immune activation, neuroaxonal injury, and clinical disease onset were modelled using linear mixed-effects models and survival analyses. In MS, EBNA-1-specific and CNS-cross-reactive IgG were elevated up to 77.8 months before diagnosis, preceding pNfL increases by 44.9 months. In NMOSD, AQP4-IgG seroconversion occurred 32.5 months before diagnosis and preceded pNfL elevations by 40.4 months. In MOGAD, pNfL elevations preceded MOG-IgG seroconversion by 11.2 months. Thus, in MS and NMOSD, humoral autoimmunity precedes detectable CNS injury, whereas in MOGAD, neuroaxonal injury occurs before circulating MOG-IgG. These distinct temporal patterns suggest differing early immunopathological trajectories and may provide a framework for future studies of early disease biology and biomarker-guided risk stratification. ### Competing Interest Statement T. Berger: has participated in meetings sponsored by and received honoraria (lectures, advisory boards, consultations) from pharmaceutical companies marketing treatments for MS: Almirall, Allergan, Bayer, Biologix, Biogen, Bionorica, BMS, Eisai, Genesis, GSK, Horizon, Janssen, Jazz Pharma, MedDay, Merck, Neuraxpharm, Newbridge, Novartis, Octapharma, Roche, Sandoz, Sanofi, Teva, TG Therapeutics and UCB. His institution has received financial support in the past 12 months by unrestricted research grants (Biogen, Bayer, BMS, Merck, Novartis, Roche, Sanofi, Teva) and for participation in clinical trials in multiple sclerosis sponsored by Alexion, Bayer, Biogen, BMS, Merck, Novartis, Roche, Sanofi, Teva. G. Bsteh: has received travel funding from Biogen, Merck, Novartis, Roche, Sanofi, and Teva; has received speaker honoraria from Biogen, BMS, Heidelberg Engineering, Janssen, Lilly, Medwhizz, Merck, Neuraxpharm, Novartis, Roche, Sanofi, Teva and Zeiss; has received honoraria for consulting Adivo Associates, Biogen, BMS, Janssen, Merck, Novartis, Roche, Sanofi and Teva. He has received unrestricted research grants from BMS, Merck and Novartis. He serves on the Executive Committee of the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS) and the Board of Directors of the International Multiple Sclerosis Visual System Consortium (IMSVISUAL). M. Breu: has received speaker honoraria from Sanofi-Genzyme. R. Hoeftberger: reports speaker honoraria from BMS and UCB. The Medical University of Vienna (Austria; employer of Dr. Hoeftberger) receives payment for antibody assays and for antibody validation experiments organized by Euroimmun (Luebeck, Germany). S. Mar: Participated in Operetta 1 and Operetta II study/Roche. No personal compensations. J.P. Nolte: has participated in meetings sponsored by, received speaker honoraria or travel funding from Novartis, Biogen, and Neuraxpharm. M. Ponleitner: has received speaker or consulting honoraria from Amicus, Sanofi-Aventis, and Novartis and participated in meetings sponsored by and received travel funding from Amicus, Merck, Novartis, and Sanofi-Genzyme, as well as grants for clinical, research, and exchange fellowships awarded by the European Academy of Neurology (EAN) and the Austrian Society of Neurology. P. Rommer: reports speaker and consultancy honoraria from A-med, Almirall, Alexion/AstraZeneca, AMGEN, Amicus, Biogen, Merck, neuraxpharm, Novartis, Roche, Sandoz, Sanofi, has received research grants from Amicus, Biogen, Merck, Roche K. Rostasy: is consultant of the Operetta II study/Roche and received honoraria for talks from Roche, Merck, Horizon, Octagam. M. Reindl: receives research support from Roche Austria. The Medical University of Innsbruck (Austria; employer of MR) receives payments for antibody assays and for antibody validation experiments organized by Euroimmun (Luebeck, Germany) The other authors declare that they have no conflict of interest. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study was approved by Institutional Review Board of the Medical University of Vienna (IRB numbers: 1123/2015, 1133/2022 1339/2022, 1668/2023, 164/2014, AN4059, #10-05039). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes De-identified data are available from the corresponding author (hannes.vietzen{at}meduniwien.ac.at) upon request and upon approval by the data-clearing committee of the Medical University of Vienna.

Rasmussen Encephalitis (RE) is a chronic, unilateral epileptic disorder mostly found in children. Neuropathologically, it is characterized by T lymphocyte infiltration targeting neurons and leading to microglia activation, astrogliosis, and cortical degeneration. Within a patient's brain, distinct pathological stages are found that offer a unique opportunity to study T cell dynamics in situ. Using quantitative multiplex fluorescence imaging, we analyzed CD103+ and CD69+ Tissue-resident memory T cells (TRM) across different disease stages. This analysis revealed that TRM were more abundant in the parenchyma than in the perivascular space, suggesting that their differentiation occurs locally after antigen encounter. Further, part of the TRM expressed Granzyme-B (GrB) and frequently were attached to neurons, suggesting that they are actively involved in neuronal destruction. While TRM showed a stage-dependent increase in older lesions, the proportions of these cells did not correlate with disease duration, indicating that their accumulation may be more dependent on the local environment in the lesion than on the length of the disease. In addition, we found that T cells using the γδ T cell receptor comprised up to 66%. Like CD8+ T cells, the γδ T cells could develop a TRM phenotype and, while expressing GrB+ granules, they were seen attached to neurons, suggesting that they are involved in neuronal destruction. Finally, analysis of exhaustion- and TRM-associated immune checkpoint control markers PD-1 and LAG-3 revealed a significant stage-dependent increase in PD-1 expression in the oldest lesions. In contrast, LAG-3 expression did not show any stage-specific pattern, pointing towards a distinct regulatory mechanism. The study demonstrates a dynamic and one-way T cell response throughout the course of RE at a given spot in the CNS: from the establishment of T cell residence after entry into the CNS, the killing of neurons, and eventually T cell exhaustion. It further suggests an important role of γδ T-cells in the propagation of disease and lesions.

OBJECTIVES:In myelin oligodendrocyte glycoprotein IgG-associated disease (MOGAD) and aquaporin-4 IgG+ neuromyelitis optica spectrum disorder (AQP4+NMOSD), the autoantibodies are mainly composed of IgG1, and complement-dependent cytotoxicity is a primary pathomechanism in AQP4+NMOSD. We aimed to evaluate the CSF complement activation in MOGAD. METHODS:CSF-C3a, CSF-C4a, CSF-C5a, and CSF-C5b-9 levels during the acute phase before treatment in patients with MOGAD (n = 12), AQP4+NMOSD (n = 11), multiple sclerosis (MS) (n = 5), and noninflammatory neurologic disease (n = 2) were measured. RESULTS:CSF-C3a and CSF-C5a levels were significantly higher in MOGAD (mean ± SD, 5,629 ± 1,079 pg/mL and 2,930 ± 435.8 pg/mL) and AQP4+NMOSD (6,017 ± 3,937 pg/mL and 2,544 ± 1,231 pg/mL) than in MS (1,507 ± 1,286 pg/mL and 193.8 ± 0.53 pg/mL). CSF-C3a, CSF-C4a, and CSF-C5a did not differ between MOGAD and AQP4+NMOSD while CSF-C5b-9 (membrane attack complex, MAC) levels were significantly lower in MOGAD (17.4 ± 27.9 ng/mL) than in AQP4+NMOSD (62.5 ± 45.1 ng/mL, p = 0.0019). Patients with MOGAD with severer attacks (Expanded Disability Status Scale [EDSS] ≥ 3.5) had higher C5b-9 levels (34.0 ± 38.4 ng/m) than those with milder attacks (EDSS ≤3.0, 0.9 ± 0.7 ng/mL, p = 0.044). DISCUSSION:The complement pathway is activated in both MOGAD and AQP4+NMOSD, but MAC formation is lower in MOGAD, particularly in those with mild attacks, than in AQP4+NMOSD. These findings may have pathogenetic and therapeutic implications in MOGAD.

Anti-glial fibrillary acidic protein (GFAP) meningoencephalomyelitis (autoimmune GFAP astrocytopathy) is a new autoimmune central nervous system (CNS) disease diagnosable by the presence of anti-GFAP autoantibodies in the cerebrospinal fluid and presents as meningoencephalomyelitis in the majority of patients. Only few neuropathological reports are available and little is known about the pathogenic mechanisms. We performed a histopathological study of two autopsies and nine CNS biopsies of patients with anti-GFAP autoantibodies and found predominantly a lymphocytic and in one autopsy case a granulomatous inflammatory phenotype. Inflammatory infiltrates were composed of B and T cells, including tissue-resident memory T cells. Although obvious astrocytic damage was absent in the GFAP-staining, we found cytotoxic T cell-mediated reactions reflected by the presence of CD8 + /perforin + /granzyme A/B + cells, polarized towards astrocytes. MHC-class-I was upregulated in reactive astrocytes of all biopsies and two autopsies but not in healthy controls. Importantly, we observed a prominent immunoreactivity of astrocytes with the complement factor C4d. Finally, we provided insight into an early phase of GFAP autoimmunity in an autopsy of a pug dog encephalitis that was characterized by marked meningoencephalitis with selective astrocytic damage with loss of GFAP and AQP4 in the lesions. Our histopathological findings indicate that a cytotoxic T cell-mediated immune reaction is present in GFAP autoimmunity. Complement C4d deposition on astrocytes could either represent the cause or consequence of astrocytic reactivity. Selective astrocytic damage is prominent in the early phase of GFAP autoimmunity in a canine autopsy case, but mild or absent in subacute and chronic stages in human disease, probably due to the high regeneration potential of astrocytes. The lymphocytic and granulomatous phenotypes might reflect different stages of lesion development or patient-specific modifications of the immune response. Future studies will be necessary to investigate possible implications of pathological subtypes for clinical disease course and therapeutic strategies.

Activation of Bruton's tyrosine kinase (BTK) has been shown to play a crucial role in the proinflammatory response of B cells and myeloid cells upon engagement with B cell, Fc, Toll-like receptor, and distinct chemokine receptors. Previous reports suggest BTK actively contributes to the pathogenesis of multiple sclerosis (MS). The BTK inhibitor Evobrutinib has been shown to reduce the numbers of gadolinium-enhancing lesions and relapses in relapsing-remitting MS patients. In vitro, BTK inhibition resulted in reduced phagocytic activity and modulated BTK-dependent inflammatory signaling of microglia and macrophages. Here, we investigated the protein expression of BTK and CD68 as well as iron accumulation in postmortem control (n = 10) and MS (n = 23) brain tissue, focusing on microglia and macrophages. MS cases encompassed active, chronic active, and inactive lesions. BTK+ and iron(+) cells positively correlated across all regions of interests and, along with CD68, revealed highest numbers in the center of active and at the rim of chronic active lesions. We then studied the effect of BTK inhibition in the human immortalized microglia-like HMC3 cell line in vitro. In particular, we loaded HMC3 cells with iron-dextran and subsequently administered the BTK inhibitor Evobrutinib. Iron treatment alone induced a proinflammatory phenotype and increased the expression of iron importers as well as the intracellular iron storage protein ferritin light chain (FTL). BTK inhibition of iron-laden cells dampened the expression of microglia-related inflammatory genes as well as iron-importers, whereas the iron-exporter ferroportin was upregulated. Our data suggest that BTK inhibition not only dampens the proinflammatory response but also reduces iron import and storage in activated microglia and macrophages with possible implications on microglial iron accumulation in chronic active lesions in MS.

Clinical, pathological, and imaging evidence in multiple sclerosis (MS) suggests that a smoldering inflammatory activity is present from the earliest stages of the disease and underlies the progression of disability, which proceeds relentlessly and independently of clinical and radiological relapses (PIRA). The complex system of pathological events driving "chronic" worsening is likely linked with the early accumulation of compartmentalized inflammation within the central nervous system as well as insufficient repair phenomena and mitochondrial failure. These mechanisms are partially lesion-independent and differ from those causing clinical relapses and the formation of new focal demyelinating lesions; they lead to neuroaxonal dysfunction and death, myelin loss, glia alterations, and finally, a neuronal network dysfunction outweighing central nervous system (CNS) compensatory mechanisms. This review aims to provide an overview of the state of the art of neuropathological, immunological, and imaging knowledge about the mechanisms underlying the smoldering disease activity, focusing on possible early biomarkers and their translation into clinical practice. ANN NEUROL 2024

Background Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease characterized by motor deficits and cognitive decline. Many immune aspects of the disease are understood through studies in the experimental autoimmune encephalomyelitis (EAE) model, including the contribution of the NF-κB transcription factor to neuroinflammation. However, the cell-specific roles of NF-κB to EAE and its cognitive comorbidities still needs further investigation. We have previously shown that the myeloid cell NF-κB plays a role in the healthy brain by exerting homeostatic regulation of neuronal excitability and synaptic plasticity and here we investigated its role in EAE. Methods We used constitutive MφIKKβΚΟ mice, in which depletion of IKKβ, the main activating kinase of NF-κB, was global to CNS and peripheral macrophages, and ΜgΙΚΚβKO mice, in which depletion was inducible and specific to CNS macrophages by 28 days after tamoxifen administration. We subjected these mice to MOG 35-55 induced EAE and cuprizone-induced demyelination. We measured pathology by immunohistochemistry, investigated molecular mechanisms by RNA sequencing analysis and studied neuronal functions by in vivo electrophysiology in awake animals. Results Global depletion of IKKβ from myeloid cells in MφIKKβΚΟ mice accelerated the onset and significantly supressed chronic EAE. Knocking out IKKβ only from CNS resident macrophages accelerated the onset and exacerbated chronic EAE, accompanied by earlier demyelination and immune cell infiltration but had no effect in cuprizone-induced demyelination. Peripheral T cell effector functions were not affected by myeloid cell deletion of IKKβ, but CNS resident mechanisms, such as microglial activation and neuronal hyperexcitability were altered from early in EAE. Lastly, depletion of myeloid cell IKKβ resulted in enhanced late long-term potentiation in EAE. Conclusions IKKβ-mediated activation of NF-κΒ in myeloid cells has opposing roles in EAE depending on the cell type and the disease stage. In CNS macrophages it is protective while in peripheral macrophages it is disease-promoting and acts mainly during chronic disease. Although clinically protective, CNS myeloid cell IKKβ deletion dysregulates neuronal excitability and synaptic plasticity in EAE. These effects of IKKβ on brain cognitive abilities deserve special consideration when therapeutic interventions that inhibit NF-κB are used in MS.

It remains elusive whether lesions and inflammation in the sub/juxtacortical white matter reflect cortical and/or meningeal pathologies. Elucidating this could have implications for MRI monitoring as sub/juxtacortical lesions are detectable by routine MRI, while cortical lesions and meningeal inflammation are not. By large-area microscopy, we quantified total and mixed active plaque loads along with densities and sizes of perivascular mononuclear infiltrates (infiltrates) in the sub/juxtacortical white matter <= 2 mm from the cortex, intra-cortically and in the meninges. Data were related to ante-mortem clinical parameters in a false discovery rate-corrected analysis. We compared 12 patients with primary progressive multiple sclerosis (PPMS) and 15 with secondary progressive MS to 22 controls. Fifteen patients and 11 controls contributed with hemispheric sections. Sections were stained with haematoxylin-eosin, for myelin and for microglia/macrophages. B cells and T cells were confirmed in a subset. Immunoglobulin G depositions in selected cortical plaques resembled depositions described before in "slowly expanding" plaques in the white matter. We quantified plaque activity by measuring microglia-dominated and macrophage-dominated areas. Sub/juxtacortical plaques (load and activity) reflected plaque activity in the cerebral cortex. Plaque activity and infiltrates were more pronounced in the sub/juxtacortical white matter than in the cerebral cortex while conversely, the total plaque load was highest in the cortex. Infiltrates correlated trans-cortically and sub/juxtacortical plaque activity reflected cortical and meningeal infiltrates. Sub/juxtacortical infiltrate sizes correlated with shorter survival after progression onset. Two patients with PPMS and putatively fatal brain stem lesions argue against incidental findings. Trans-cortical inflammatory flares and plaque activity may be pathogenic in progressive MS. We suggest emphasis on sub/juxtacortical MRI lesions as plausible surrogates for cortical and meningeal pathologies and, when present, as indicators for cognitive testing.

Myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD) is an immune-mediated demyelinating disease that is challenging to differentiate from multiple sclerosis (MS), as the clinical phenotypes overlap, and people with MOGAD can fulfil the current MRI-based diagnostic criteria for MS. In addition, the MOG antibody assays that are an essential component of MOGAD diagnosis are not standardized. Accurate diagnosis of MOGAD is crucial because the treatments and long-term prognosis differ from those for MS. This Expert Recommendation summarizes the outcomes from a Magnetic Resonance Imaging in MS workshop held in Oxford, UK in May 2022, in which MS and MOGAD experts reflected on the pathology and clinical features of these disorders, the contributions of MRI to their diagnosis and the clinical use of the MOG antibody assay. We also critically reviewed the literature to assess the validity of distinctive imaging features in the current MS and MOGAD criteria. We conclude that dedicated orbital and spinal cord imaging (with axial slices) can inform MOGAD diagnosis and also illuminate differential diagnoses. We provide practical guidance to neurologists and neuroradiologists on how to navigate the current MOGAD and MS criteria. We suggest a strategy that includes useful imaging discriminators on standard clinical MRI and discuss imaging features detected by non-conventional MRI sequences that demonstrate promise in differentiating these two disorders.