Neurology Neuroimmunology & Neuroinflammation

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.

Background: Progressive multiple sclerosis (MS) is characterized by ongoing neurodegeneration and limited therapeutic options. Circulating metabolites provide insight into disease biology, yet biomarkers that predict disability progression and reflect treatment response are lacking. We aimed to identify metabolomic signatures associated with longitudinal MRI measures of brain atrophy and to evaluate whether ibudilast treatment was associated with metabolite trajectories over time. Methods: We repeatedly profiled 1,726 plasma metabolites using untargeted UPLC-MS/MS in 244 participants from the 96-week SPRINT-MS randomized trial of oral ibudilast, up to 100 mg daily, versus placebo. Weighted gene co-expression network analysis was used to derive groups of related metabolites. Associations between baseline metabolite groups and longitudinal MRI outcomes were evaluated using linear mixed-effects models adjusted for demographic, clinical, and treatment covariates. The primary outcome was the rate of whole-brain atrophy measured by brain parenchymal fraction (BPF), defined as the proportion of intracranial volume occupied by brain tissue. Secondary outcomes included white matter fraction (WMF), gray matter fraction (GMF), and cortical thickness (CTH). Metabolite groups nominally associated with MRI outcomes, defined as p < 0.05, were followed by individual metabolite analyses to identify potential drivers. Significant metabolites were tested for replication in a comparable real-world observational HEAL-MS cohort with longitudinal MRI data. Lastly, we tested whether ibudilast treatment was associated with metabolite trajectories and performed metabolite set enrichment analysis. Findings: Higher baseline levels of glycerophospholipids were associated with slower decline in both BPF and WMF, and sphingomyelins were similarly associated with slower BPF decline. For example, higher 1-palmityl-2-stearoyl-GPC (O-16:0/18:0) levels were associated with slower BPF decline in SPRINT-MS (beta = 0.016 [95% CI: 0.008, 0.024]; p = 4.35 x 10^-5) and replicated in HEAL-MS (beta = 0.108 [95% CI: 0.006, 0.211]; p = 3.90 x 10^-2). Metabolites associated with GMF preservation were enriched in androgenic steroids and steroid sulfates, with consistent positive associations observed in the replication cohort, whereas metabolites inversely associated with CTH were predominantly xenobiotic-related. Ibudilast treatment was associated with increased sphingomyelin species, such as palmitoyl sphingomyelin (d18:1/16:0; beta = 0.185 [95% CI: 0.085, 0.286]; FDR = 1.79 x 10^-2), and decreased levels of amino acid-related metabolites, such as anthranilate (beta = -0.270 [95% CI: -0.403, -0.137]; FDR = 3.87 x 10^-2). Pathway-based analyses corroborated these findings, highlighting glycerophospholipid and sphingolipid metabolism as key pathways implicated in brain atrophy in MS. Interpretation: Distinct lipid subsets were associated with slower brain atrophy in people with MS, and ibudilast treatment was associated with metabolite alterations in potentially neuroprotective directions. Metabolomics may provide prognostic and pharmacodynamic biomarkers for progressive MS.

BackgroundExtracellular matrix (ECM) remodeling is increasingly recognized as an important component of neuroinflammatory pathology in multiple sclerosis (MS), yet the mechanisms by which CNS cells sense and respond to alterations in their mechanical environment and the spatial across which mechanical changes can influence cellular behavior remain poorly understood. Piezo1 is a mechanosensitive ion channel that regulates cellular responses to mechanical stimuli and has recently emerged as a potential modulator of neuroinflammation. MethodsExperimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 wildtype mice using myelin oligodendrocyte glycoprotein (MOG):35-55. Immunohistochemical analyses were performed in spinal cord gray matter (GM), normal-appearing white matter (NAWM), and white matter lesion (LES) regions to assess ECM remodeling, total Piezo1 expression, and astrocyte-specific Piezo1 expression during acute and chronic EAE stages. Correlations with clinical EAE severity were determined. In parallel, mixed primary murine glial cultures were exposed to substrates of different stiffness and analyzed by transcriptomic profiling to investigate mechanobiological responses in vitro. ResultsECM-associated proteins, including glial fibrillary acidic protein (GFAP), fibronectin-1 and matrix metalloproteinase-3 (MMP3), were regionally upregulated during EAE, indicating widespread tissue remodeling beyond focal inflammatory lesions. Total Piezo1 expression was increased within lesions and transiently elevated in GM, whereas astrocyte-specific Piezo1 remained persistently upregulated during both acute and chronic EAE. Astrocytic Piezo1 expression correlated closely with ECM remodeling and clinical EAE severity, particularly in GM and NAWM. Notably, both total and astrocyte-specific Piezo1 showed stronger associations with clinical disability than classical inflammatory markers. Transcriptomic analysis revealed pronounced stiffness-dependent responses in glial cells, including alterations in extracellular matrix organization, cytokine signaling, cell adhesion, and proliferative pathways. ConclusionsOur findings identify astrocytic Piezo1 as a prominent component of neuroinflammatory tissue remodeling during EAE. The close association of Piezo1 with ECM alterations, clinical disease severity, and stiffness-dependent glial responses supports a link between neuroinflammation and mechanosensory signaling. These results highlight mechanosensation as a potentially important contributor to CNS pathology and establish Piezo1 alteration as a candidate biomarker for neuroinflammatory disease.

Background and ObjectivesCortical lesions are common in multiple sclerosis (MS) and associated with disability, but their characterization in early MS has been limited. Here, we aimed to characterize cortical lesions in newly diagnosed MS with 7 tesla (T) brain MRI. MethodsAdults within 14 months of relapsing-remitting MS diagnosis underwent 7T brain MRI and clinical evaluation at Mount Sinai. Cortical lesions were identified using T1-weighted (w) (median of three acquisitions) and T2*w images (both at 0.5mm3). Non-cortical brain lesions were segmented on 0.7mm3 T1w images. Lesion burden in newly diagnosed MS was compared with a previously analyzed NIH cohort with longer time since diagnosis, imaged using a similar protocol. Results61 individuals were included in the newly diagnosed MS cohort (mean age 34 {+/-} 4 years; 72% female; median time since diagnosis 5 months, interquartile range [IQR] 6). Cortical lesions were identified in 50/61 (81%) individuals, and subpial cortical lesions were identified in 46 (75%). Median cortical lesion number was 5 (IQR 11), median volume 319 l (IQR 1049). Cortical lesions constituted a median of 14% of total brain lesion volume (IQR 43%), and in 21% of individuals, cortical lesions constituted >50% of total brain lesion volume. Cortical lesion number was associated with worse 9-hole peg test ({rho}=0.33, p=0.008) and Symbol Digit Modalities Test performance ({rho}=-0.29, p=0.02). When pooled with the NIH cohort (n=60, median time since diagnosis 12 years, IQR 17), non-cortical lesion volume was [~]3.5 times higher in people with time since diagnosis >36 months (median 4.7 ml, IQR 8.7) vs [&le;]36 months (median 1.2 ml, IQR 2.4, p<0.001). In contrast, cortical lesion volume was only [~]1.3 times higher in people with time since diagnosis >36 months (median 416 l, IQR 1013) vs [&le;]36 months (median 318 l, IQR 925, p=0.04). Non-cortical lesion volume was moderately associated with time since diagnosis ({rho}=0.54, p<0.001) vs {rho}=0.27 (p<0.001) for cortical lesions. DiscussionCortical lesions are prevalent in newly diagnosed MS and constitute a substantial portion of total lesion burden. Cortical lesion volume is similar in early vs established MS, suggesting most cortical lesions form early in disease.

Background: Comorbidities are common in multiple sclerosis (MS) and may influence disability outcomes, but their dynamic impact on bidirectional disability transitions and long-term disability remains incompletely understood. Better understanding of this longitudinal relationship could inform personalized disability management strategies for people with MS. Methods: We leveraged two large electronic health record (EHR)-linked MS registries and applied multi-state Markov models (MSMs) to examine the extent to which individual comorbidities and overall comorbidity burden were associated with short-term disability transitions, long-term disability transition probabilities, and expected time spent in each disability state. We additionally compared MSM-based predictions of confirmed disability worsening (CDW) with Cox proportional hazards (CoxPH) model-based predictions using the integrated Brier score with bootstrap validation. Results: Among 3,723 patients with MS (74.6% female; 86.2% non-Hispanic White; mean age=41.9 years; mean disease duration=5.4 years) contributing 41,860 disability assessments over a mean follow-up of 7.3 years, higher cardiometabolic and psychiatric comorbidity burden was associated with increased transition intensity toward worse disability states and decreased transition intensity toward improvement, with a stepwise gradient across burden levels. Compared with patients without comorbidities, those with [&ge;]4 comorbidities had a 28% higher risk of worsening (HR=1.28 [1.06, 1.55]) and a 20% lower risk of improvement (HR=0.80 [0.67, 0.95]). Each individual comorbidity was significantly associated with worse disability transitions. Long-term estimates indicated a higher 5-year probability of severe disability and fewer years spent in the no-disability state among patients with greater comorbidity burden. CoxPH models showed directionally consistent associations but lower predictive accuracy for CDW compared with MSMs. Conclusion: Cardiometabolic and psychiatric comorbidities are associated with worse disability trajectories in MS, reducing improvement and accelerating progression. By providing a nuanced framework to quantify short-term disability transitions and long-term disability patterns, MSMs may have real-world clinical utility in disability prediction.

Introduction- Choroid plexus (CP) enlargement on brain MRI has been identified as an emerging neuroinflammatory biomarker in multiple sclerosis (MS), yet its relationship to downstream parenchymal neurochemical abnormalities remains unknown. Proton magnetic resonance spectroscopy (1H MRS) enables non-invasive in vivo quantification of neurometabolites, making it well-suited to probe downstream consequences of CP pathology in MS. Methods- Ultra-high-field 7T 1H MRS was performed in 45 people with MS (pwMS) (28 Relapsing Remitting MS, RRMS; 17 Progressive MS, PMS) and 43 age- and sex-matched healthy controls (HCs) in the posterior cingulate cortex (PCC) and centrum semiovale white matter (CSWM). CP volume, EDSS, and MS Functional Composite measures were also acquired. Group differences in metabolite concentrations were evaluated using Mann-Whitney U tests with correction for multiple comparisons, and associations between CP volume, altered metabolites, and clinical disability and functional measures were investigated. Results- Myo-inositol (mI) was significantly elevated and total N-acetylaspartate was reduced in both MS subtypes, in the CSWM. In PMS, CP volume was positively associated with CSWM mI/total creatine (tCr) ({rho} = 0.63, p = 0.008), an association absent in RRMS. Across the combined MS cohort, CP volume correlated significantly with EDSS ({rho} = 0.40, p = 0.006). Conclusions- WM mI/tCr was elevated and tNAA/tCr was reduced across MS phenotypes compared with controls, reflecting a dual metabolic signature consistent with concurrent glial overactivation and neuroaxonal compromise. Increased CP volume was associated with greater neurological disability across MS phenotypes. The association of CP enlargement with CSWM mI/tCr in PMS suggests a potential link between CP-mediated periventricular inflammation and progressive WM glial pathology. Collectively, these findings support CP volume as a clinically relevant, non-invasive biomarker and restoring CP integrity as a potential therapeutic target in PMS, where effective treatments remain limited.

The course of multiple sclerosis (MS) is highly heterogeneous, yet the biological mechanisms underlying this variability remain incompletely understood. Although metabolic alterations have increasingly been associated with disease progression, existing observational evidence is limited by confounding, reverse causation, and an inability to establish causal mechanisms. To bridge this gap, we used a metabolome-wide Mendelian Randomization (MR) framework, including thorough sensitivity analyses, to identify metabolites genetically linked to MS severity that can causally affect it. Bidirectional MR analyses revealed a subset of amino acid and lipid pathways with strong, consistent effects across different MR approaches, confirmed by tests for heterogeneity, horizontal pleiotropy, and LD confounding. For metabolites prioritized by metabolome-wide MR with evidence of causal effects, we conducted genetic colocalization at loci encompassing proximal enzyme-encoding genes, leveraging the corresponding instrumental variants to assess shared underlying genetic signals. This process revealed shared genetic signals between metabolite levels and MS severity, mapped to the FADS1/2 and CYP4F2 loci. A subsequent pathway-resolved set of cis-MR analyses across FADS1/2-derived polyunsaturated fatty acid (PUFA) metabolites, using a functional variant that proxies reduced {triangleup}5-desaturase activity, showed consistent effects indicating that FADS1 perturbation is associated with MS severity. Collectively, these results highlight FADS1 as a key driver of PUFA-related causal effects on MS severity in both systemic (circulating metabolites) and brain cell-specific contexts. Additional supportive cis-MR evidence implicates the disruption of CYP4F2 as another PUFA-metabolizing enzyme.

Importance: As new treatments increase quality and length of life in people with multiple sclerosis (MS), effective prevention and management of common comorbidities, including Diabetes Mellitus (DM), is increasingly important. Objective: To compare incidence of DM and its associations with hospitalisation and mortality in adults with MS and matched controls. Design: Using English primary care data from the Clinical Practice Research Datalink (CPRD), linked to Hospital Episode Statistics and national mortality records, we matched adults with MS diagnosed between 2000 and 2023, with up to ten controls without MS by age, sex, and practice. We excluded individuals with preexisting DM, defined using diagnostic and management codes. Outcomes included all-cause hospitalisation (number and duration) and mortality. We used Poisson, negative binomial, linear, and Cox proportional hazards models, adjusting for demographic and socioeconomic factors, adding interaction terms to examine if ethnicity, deprivation, and urbanity were associated with outcomes. Results: We included 9,010 individuals with MS and 78,121 matched controls. Over a mean follow-up of 13.2 years, people with MS had over twice the incidence of DM compared with controls (adjusted incidence rate ratio [aIRR]=2.26, 95% CI: 1.96 to 2.61, p<0.001). Among people with MS, incident DM was associated with higher hospitalisation rates (aIRR=1.82, 95%CI: 1.47 to 2.28, p<0.001), longer hospitalisation duration (median 18 vs 4 days, adjusted beta;=0.53, 95%CI: 0.41 to 0.65, p<0.001), and increased all-cause mortality when incident DM was modelled as a time-varying exposure (adjusted hazard ratio=1.46, 95%CI: 1.17 to 1.82, p<0.001), compared to those who did not develop DM. Similar patterns were observed among controls (hospitalisation rates: aIRR = 2.96, 95% CI 2.63 to 3.23, p<0.001; hospitalisation duration: adjusted {beta} = 0.93, 95% CI: 0.86 to 0.99, p<0.001; mortality [time-varying]: HR = 1.50, 95% CI: 1.27 to 1.77, p<0.001). The relationship between DM and increased hospitalisation was stronger in rural areas among those with MS and stronger in White groups among controls. Conclusions: People with MS are more likely to be diagnosed with DM, resulting in greater all-cause hospitalisation and all-cause mortality. This highlights the importance of equitable screening, prevention, and management of DM in people living with MS, with particular attention to geographical health inequalities.

Background: Serum neurofilament light chain (NfL) indexes axonal injury and glial fibrillary acidic protein (GFAP) astrocytic pathology in multiple sclerosis (MS). GFAP rises disproportionately as relapsing-remitting MS (RRMS) shifts to progressive forms on research-grade SIMOA. The commercial Roche Elecsys ECLIA platform reads six-fold lower and is undescribed across subtypes. Objective: To describe both markers by MS subtype on ECLIA. Methods: Retrospective single-center analysis of 603 MS patients (2022-2026). NfL and GFAP were measured by LabCorp Roche Elecsys ECLIA; subtype came from ICD-10 codes and notes. We examined both markers by subtype, their correlation, and NfL against gadolinium-enhancing (Gd+) MRI lesions. Results: Median NfL was 1.32 pg/mL (IQR 1.01-1.91). Both rose with stage, steeper for GFAP: NfL 1.18 (RRMS), 1.54 (SPMS, p<0.001), 1.78 (PPMS, p=0.001); GFAP 41.90, 63.80 (p<0.0001), 75.75 (p=0.08, n=6). SPMS and PPMS GFAP did not differ (p=0.83). The markers correlated moderately (r=0.569). Of 34 Gd+ encounters with NfL within 30 days, 3 (9%) were elevated. Conclusion: On ECLIA, both markers rose with MS stage, GFAP more steeply, and both progressive subtypes exceeded RRMS. NfL rarely flagged a recent Gd+ lesion, consistent with its delayed kinetics. The two index distinct processes and reproduce on an orderable assay a profile once confined to research-grade SIMOA.

Comprehensive biomarkers of multiple sclerosis (MS) capable of simultaneously diagnosing the condition, capturing symptom severity and predicting treatment efficacy remain elusive. Although several studies have highlighted the pivotal role played by demyelinating lesions in determining MS structural pathology, their relationship with symptom severity is limited. Here, we combined personalized computational brain modeling with magnetoencephalography (MEG) recordings from 17 MS patients and 20 healthy controls (CTR) to derive personalized brain network excitability parameters, which we tested as MS biomarkers. Personalized parameters discriminated between CTR and MS participants with high accuracy, also classifying between progressing and remitting MS patients. Notably, they also predicted MS clinical scales across multiple domains. In all clinical tasks, personalized parameters consistently outperformed standard clinical measures and total lesion loads. Together, these results highlight the potential of personalized brain modelling in deriving integrative MS biomarkers, capable of simultaneously identifying the condition, classifying MS subtypes and predicting symptom severity. d brain modelling in deriving integrative MS biomarkers, capable of simultaneously identifying the condition, classifying between MS subtypes and predicting the severity of symptomatology.

Neurodegeneration is a major determinant of disability progression in multiple sclerosis (MS), yet the pathophysiological mechanisms associating inflammation to neuronal insult remain poorly understood. We recently identified Dendritic Syntaphilin Intrusion (DSI), a novel excitoxicity pathway in which the axonal mitochondrial anchor syntaphilin (SNPH) aberrantly translocates into dendrites, causing neurodegeneration in a non-inflammatory model of MS. However, whether this protein intrudes abruptly into dendrites in inflammatory MS pathology is still not clear. Here, we investigated the role of synaptic and extrasynaptic NMDA receptors (NMDAR) in regulating the intrusion of Syntaphilin into dendrites. Using primary hippocampal neuronal cultures, we examined how the balance between synaptic GluN2A-containing and extrasynaptic GluN2B-containing NMDARs influences DSI under inflammatory conditions. Pharmacological and viral-mediated approaches were employed to manipulate NMDAR subtype activity and evaluate their impact on DSI. Inflammatory cytokines discernibly sensitized neurons to DSI. Our results revealed that blockade of synaptic NMDARs significantly increased DSI, whereas inhibition of extrasynaptic NMDARs reduced DSI. These findings demonstrate opposing roles of NMDAR subtypes, with GluN2A-containing synaptic receptors inhibiting DSI and fostering neuronal survival, while GluN2B-containing extrasynaptic receptors enhancing DSI and neurodegenerative signaling. Manipulation of the GluN2A/GluN2B balance showed opposite effect on DSI, suggesting a relationship between NMDAR subtype signaling and SNPH mislocalization. Overall, our findings extend the relevance of DSI from non-inflammatory MS to inflammatory MS and identify DSI as a downstream convergence point linking inflammatory cytokines and excitotoxic NMDAR signaling to neuronal insult. These results reveal DSI as a potential mechanistic link between inflammatory signaling and excitotoxic neuronal injury and indicate that modulation of GluN2B-dependent pathways warrants further investigation in inflammatory neurodegenerative disorders.

Introduction: Incomplete recovery from relapses contributes to long-term disability accumulation in relapsing remitting multiple sclerosis (RRMS), yet the relationship between immune regulation and relapse recovery remains poorly defined. Objective: To longitudinally characterize regulatory/effector immune cell dynamics in untreated RRMS patients and assess their association with immune balance and relapse recovery. Methods: Monocytic myeloid-derived suppressor cells (M MDSCs), regulatory T cells (Treg), and effector CD4 T cell subsets were measured in blood from 69 untreated RRMS patients sampled during relapse or remission and reevaluated after 12 months. Associations with clinical recovery after relapse were examined. Results: During relapse, patients exhibited higher M MDSC and Treg frequencies than in remission, while effector T cell subsets remained unchanged. Over one year, M-MDSCs increased consistently regardless of baseline clinical status, whereas Treg frequencies remained stable. Effector to M MDSC ratios were markedly elevated during relapse and declined over time, while effector-to-Treg ratios showed minimal variation. M MDSC levels during relapse were associated with sustained regulatory features at 12 month follow up. Importantly, higher baseline M MDSC levels, but not Treg frequencies, were associated with complete relapse recovery at one year. Conclusion: These findings suggest that circulating M-MDSCs, but not Treg, reflect interindividual differences in immune regulation and clinical recovery after relapse in early RRMS.

Introduction: Progressive multiple sclerosis (MS), including primary progressive MS (PPMS) and non-active secondary progressive MS (nSPMS), remains an unmet need, as few treatments target innate immune pathways. Masitinib (AB1010) is a selective tyrosine kinase inhibitor that targets c-Kit and colony-stimulating factor 1 receptor pathways. This mechanism disrupts mast cell-microglia interactions, key innate immune effectors in progressive MS pathogenesis, reducing neuroinflammation and neuronal damage. In the phase 3 AB07002 trial, masitinib (4.5 mg/kg/d) over 96 weeks met its primary endpoint. Comparable signals in PPMS and nSPMS indicated masitinib benefited both phenotypes. Secondary analyses showed that masitinib lowered the progression to wheelchair dependence (EDSS [&ge;]7, 12 weeks) and reduced the 12-week confirmed EDSS progression risk by 37% versus placebo, although the results were underpowered for these endpoints. Methods: This study aimed to confirm that oral masitinib achieves central nervous system (CNS) concentrations sufficient to modulate CSF1R and wild-type c-Kit, thereby underpinning its neuroprotective potential. Male Sprague Dawley rats (n=12, ~200 g) were administered a single oral dose (30 mg/kg). Plasma and brain samples were collected at 2, 4, 8, and 24 hours post-dose (n=3 per time point). Masitinib (AB1010) and its metabolite (AB3280) were quantified in plasma and brain homogenates using LC-MS/MS. Results: Masitinib reached a brain Cmax of 223.5 ng/mL (~450 nM), exceeding IC50 values for CSF1R and wild-type c-KIT by ~5-fold and 2-fold, respectively, indicating effective CNS target engagement. The active metabolite AB3280 also achieved brain Cmax levels with full inhibitory activity. Masitinib demonstrated consistent CNS penetration supported by a proportional plasma-to-brain exposure relationship. Conclusion: The favorable CNS penetration and safety profile of masitinib, alongside its unique mast cell inhibition, position it as a compelling candidate for progressive MS treatment, either as monotherapy or in combination with other agents. This multifaceted immunomodulatory approach addresses critical unmet needs in progressive MS and supports further clinical development.

Guillain-Barre syndrome is an acute immune-mediated polyradiculoneuropathy with heterogeneous outcomes and limited molecular biomarkers for diagnosis, disease monitoring, and prognosis. To elucidate the circulating proteomic profile of this disorder and identify candidate biomarkers associated with disease activity and recovery, we measured over 6,500 proteins using an aptamer-based proteomic platform. We analysed paired, longitudinal sera from 20 patients at disease onset and one-year follow-up, alongside 15 healthy controls. Unbiased differential protein abundance and gene-set enrichment analyses were performed. Candidate proteins were validated using conventional immunoassays in a cohort including healthy and disease controls. We identified 39 differentially abundant proteins between the acute and recovery phases and 248 proteins altered in acute Guillain-Barre syndrome compared to controls. The acute phase was characterised by a marked enrichment in systemic immune cascades and muscle sarcomere proteins, alongside a significant depletion of axonal adhesion molecules. Serum amyloid A1 (SAA1) emerged as the most strongly increased protein in the acute phase. Validation through independent immunoassays confirmed robust serum amyloid A elevations at disease onset relative to the one-year recovery phase, healthy controls, and relevant post-infectious and neuromuscular disease controls (acute disseminated encephalomyelitis and myasthenia gravis), underscoring a peripheral nerve-specific inflammatory response. Furthermore, unexpected elevations of cardiac troponin T (cTnT) were observed at disease onset. Clinical validation using high-sensitivity assays demonstrated that cTnT exceeded the diagnostic 99th percentile upper reference limit in 25.5% of acute Guillain-Barre syndrome patients. A similarly high frequency of elevation in the myasthenia gravis disease control group (42.1%) suggests these increases predominantly reflect neuromuscular damage rather than myocardial injury. Finally, Mendelian randomisation provided causal genetic evidence linking specific systemic proteins to disease susceptibility, identifying robust roles for SERPING1 (plasma protease C1 inhibitor), CNDP1 (an antioxidant protein), and CRISPLD2 (a lipopolysaccharide-binding protein that regulates endotoxin function). Together, this comprehensive proteomic characterisation reveals distinct, stage-specific molecular signatures in Guillain-Barre syndrome. Importantly, it suggests SAA1 as a robust marker of acute peripheral nerve inflammation and challenges the conventional interpretation of elevated cTnT in severe neuropathies and neuromuscular disorders. Furthermore, this work provides a novel dataset to explore future targeted therapeutic development in Guillain-Barre syndrome.

Introduction Current treatments for multiple sclerosis (MS) do not address the pathological processes of neurodegeneration and chronic demyelination. This, coupled with the significant challenges of translating promising phase 2 results to phase 3 trial success, highlights the need for more efficient trial designs, such as platform multi-arm multi-stage (MAMS) trial approaches. MAMS trials have demonstrated success in areas such as oncology and infectious diseases. They are typified by a statistically robust core trial design that allows the addition of further treatment arms and utilisation of interim outcome analyses at pre-defined timepoints, to determine whether to terminate a treatment arm early or proceed to the final outcome analysis. To address the challenges in progressive multiple sclerosis (PMS) treatment discovery, the Optimal Clinical Trials Platform for PMS (OCTOPUS) trial was developed. It currently utilises MRI whole-brain atrophy as its interim outcome measure and the clinically relevant composite Expanded Disability Status Scale Plus (EDSS-Plus) as its final outcome measure. A rigorous and systematic drug selection process that assessed preclinical in vitro and animal model evidence, along with additional human data, led to the prioritisation of R/S-alpha lipoic acid (R/S-ALA) and metformin for testing against placebo, targeting pathobiological mechanisms relevant to PMS. All participants will be eligible to receive the current standard of care, including disease-modifying treatments (DMTs). Method and analysis OCTOPUS will be a multi-centre, randomised, placebo-controlled, double-blind, phase 3, MAMS trial of participants aged 25 to 70 years (inclusive) with PMS and an EDSS score of 4.0 to 8.0 (inclusive). Steady progression must be the major cause of increasing disability rather than relapse in the preceding 2 years. In the trial s first candidate drug cycle, participants will be allocated to R/S-ALA, metformin, or placebo in a 1:1:1 ratio. Cycle 1 active treatments will start as R/S-ALA 600 mg once daily, increased after 4 weeks to 600 mg twice daily, or metformin 1 g once daily, increased after 4 weeks to 1 g twice daily. The trial will be multinational, with participation from 28 hospitals across the UK and 10 hospitals in Australia. Clinician-reported measures will include: the EDSS-Plus and the individual components: EDSS, Timed 25 Foot Walk (T25FW); 9 Hole Peg Test (9HPT); Symbol Digit Modalities Test (SDMT); Sloan Low Contrast Visual Acuity (SLCVA); and Relapse assessment. Patient-reported outcomes include MS specific walking, fatigue, pain, and impact scales. We will include a health economic analysis. Analysis stage 1 will require randomisation of 125 participants per arm and utilise MRI percentage brain volume change (PBVC) with the Structural Image Evaluation using Normalisation of Atrophy (SIENA) technique from baseline to 78 weeks. A positive outcome in analysis stage 1 will detect a 0.15% per year whole brain atrophy difference with a one-sided alpha of 0.35 and power of 95%, ensuring a low probability of erroneously rejecting a treatment arm at this stage. Any arms that show a positive effect will proceed to final analysis stage 2. Analysis stage 2 will require 600 participants per arm. Participants included in stage 1 will also be included in the stage 2. Analysis stage 2 will evaluate time to 6-month confirmed disability progression in the EDSS-Plus, in order to detect a 25% hazard ratio reduction with 90% power and an alpha of 0.05. Assuming one treatment arm proceeds to analysis stage 2, the trial will recruit approximately 1,200 participants and last about 6 years. This is approximately two-thirds the size and half the duration of separately conducted two-arm phase 2 and 3 trials. Ethics and dissemination The protocol was approved by the London Hampstead REC (22/LO/0622). This manuscript is based on protocol version 8.0, 28th August 2025. The findings of this trial will be disseminated through peer-reviewed publications and conference presentations. There will be a close communication strategy developed with the UK MS Society (MSS) and full patient and public involvement and engagement (PPIE). Trial registration ISRCTN: 14048364 EudraCT number: 2021-003034-37 CTA 20363/0445 IRAS number: 1003943 Secondary identifying numbers: ND001, CPMS 54274 Strengths and limitations - The OCTOPUS trial will be the first platform multi-arm multi-stage phase 3 trial in PMS, offering the potential to significantly expedite clinical trial processes with advantages in cost- and time-efficiency, focusing specifically on the poorly treated pathobiological processes of chronic neurodegeneration and demyelination - It will begin by assessing two promising drug candidates, immediate-release metformin and R/S-ALA, and will expand over the duration of the trial to include more drug arms under the same trial master protocol - The flexible and statistically robust trial design means that several components of the design (such as the early analysis stage 1 interim outcome) can be updated in line with evolving scientific knowledge - It will ultimately be the largest ever investigator-initiated phase 3 trial in PMS - It will include a range of national and international trial sites, including neuroscience centres and district general hospitals - It will have a high inclusion limit for age (up to 70 years) and disability (up to EDSS 8.0) - Several components (the telephone EDSS and virtual patient-reported outcome measures) will be amenable to remote collection increasing inclusivity and thus addressing public and participant suggestions, while minimising the risk of missing data - The main challenges in this trial design are the statistical and methodological complexity involved in design and implementation, and interpretation of interim trial results. Conclusion The trial launched cycle 1 in January 2023. Analysis stage 1 recruitment of 375 participants was achieved in November 2024, enabling planned interim analysis stage 1 to be conducted by late 2026 (Figure 1). On the 1st of June 2026, in the UK, 24 sites are active with a further 4 in set-up as part of stage 2, and in the Australian extension, Platform Adaptive Trial for Remyelination and Neuroprotection in Multiple Sclerosis (PLATYPUS), 1 site is active, with 9 additional sites in set-up.

The human endogenous retrovirus type W (HERV-W) has been identified as a human-specific neuropathological factor that preferentially affects glial cell types in multiple sclerosis (MS). Recent work using transgenic mice with expression of the HERV-W envelope (ENV) protein unveiled that this endogenous retroviral element disrupts myelin repair and polarizes microglial and astroglial cells towards axon-damaging neurotoxic phenotypes. Moreover, initial clinical trials using Temelimab, a neutralizing antibody targeting the HERV-W ENV protein, have provided circumstantial evidence that ENV exerts anti-regenerative and neurodegenerative effects in MS patients. Aligning these observations, it was therefore concluded that HERV-W represents an important factor contributing to disease progression independent of relapse activity (PIRA). Building on these findings, we here applied a neutralizing anti-ENV antibody in a non-inflammatory demyelination mouse model to directly evaluate its potential to mitigate neurodegeneration and ameliorate remyelination. In transgenic mice with human-specific expression of the HERV-W ENV protein, repetitive intraperitoneal anti-ENV antibody injections resulted in accelerated oligodendroglial differentiation, enhanced remyelination, axonal protection, and reduced neurofilament light chain leakage in the serum. Neurotoxic microglial traits were also reduced, while homeostatic parameters were stabilized. As astroglial cells underwent a similar shift, inducing regenerative traits at the expense of toxic parameters, anti-ENV application overall generated a less hostile cellular environment. This study provides direct evidence of the capacity of HERV-W neutralizing antibodies to access the central nervous system and to ameliorate damage conferred by this viral entity previously associated with smouldering disease processes. Significance StatementAlthough neurodegeneration is a hallmark of multiple sclerosis (MS), its underlying mechanisms are poorly understood. Clinically, it manifests as smouldering MS or progression without relapse activity (PIRA). This is the primary factor leading to the accumulation of clinical disability and is currently untreatable. This study provides the first direct evidence that antibodies directed against the HERV-W ENV protein can attenuate the activity of neurodegeneration-promoting glial cells in vivo. Our data validates neutralization of this endogenous retroviral element as a promising therapeutic approach particularly relevant to the chronic form of MS.

ImportanceIn multiple sclerosis (MS), high-efficacy disease-modifying therapies (HEDMTs) effectively control relapse-associated worsening (RAW), but progression independent of relapse activity (PIRA) remains inadequately addressed. As HEDMTs become the standard of care, developing new therapies that target this residual progression is a critical unmet need. ObjectiveThis study quantifies disability progression in MS patients treated with ocrelizumab to evaluate how confirmed EDSS disability progression (EDSS-CDP) would perform as an endpoint in future trials using HEDMT as comparators. DesignRetrospective longitudinal cohort study. SettingPooled dataset from four multicenter phase III and IV clinical trials. Participants1,859 people with (pw) relapsing MS (RMS), primary progressive MS (PPMS), and secondary progressive MS (SPMS) who were treated with ocrelizumab within the OPERA I/II, ORATORIO, and CONSONANCE trials. InterventionOcrelizumab. Main Outcomes and MeasuresWe developed a hierarchical Bayesian model to analyze longitudinal EDSS trajectories using two components: an offset effect, used to capture changes occurring rapidly after treatment onset, followed by a steady, long term linear progression over time. We used this model to simulate future clinical trial scenarios, assuming different drug effects on the offset and the long term linear progression. ResultsOur model accurately describes longitudinal EDSS changes and the risk of EDSS-CDP in ocrelizumab-treated subjects. Disability improvement (offset effect) was most prominent in pwRMS, while pwPPMS exhibited the highest long-term progression rates. Baseline T1 gadolinium-enhancing lesions were associated with a greater initial benefit. Simulations of typical phase III trials suggest that the hazard ratio on the EDSS-CDP endpoint is mostly influenced by the magnitude of the offset effect rather than the impact on long-term linear progression. Conclusions and RelevanceWe attribute the disability improvement observed shortly after treatment onset to resolving focal inflammation, and the long-term steady progression rate to disease mechanisms not fully addressed by ocrelizumab. Our simulation results show that within the current trial paradigm, which uses EDSS-CDP as a measure of disability progression, the ability of a treatment to induce an initial improvement is the primary determinant of success. These results emphasize the urgent need for both innovative clinical trial designs and more sensitive endpoints to adequately assess the next generation of MS therapies targeting gradual disability progression. Key PointsO_ST_ABSQuestionC_ST_ABSWill the standard multiple sclerosis disability progression endpoint, confirmed EDSS disability progression (EDSS-CDP), prove to be an accurate measure of the efficacy of new therapies addressing long-term progression when compared against high-efficacy treatments (HET)? FindingsIn this modeling study of 10-year ocrelizumab data, observed changes in EDSS were characterized by an early improvement followed by a linear long-term worsening. EDSS-CDP was shown to be highly sensitive to initial improvement. Since this phenomenon strongly influences the overall treatment effect, trials that use ocrelizumab, or similar HET as a comparator may fail to identify novel treatments designed to further slow long-term progression. MeaningCurrent trial designs may be inadequate for evaluating next-generation MS therapies, necessitating the development of better metrics to capture treatment effects on gradual progression.

Background Social and geographic barriers contribute to worse outcomes in patients with multiple sclerosis (MS) and related disorders, but these factors remain poorly characterized among Latino patients. We evaluated associations between distance to specialty care, neighborhood deprivation, insurance status, and clinical outcomes among Latinos with MS and related disorders. Methods We conducted a retrospective study of Latino adults with MS, neuromyelitis optica spectrum disorder, and myelin oligodendrocyte glycoprotein antibody-associated disease. Demographic, clinical, and socioeconomic variables were abstracted from the medical record. Distance to care was defined as residence [&ge;]50 vs. <50 miles from clinic and neighborhood deprivation as Area Deprivation Index (ADI) state rank. We used unadjusted and multivariable regression to evaluate associations with Expanded Disability Status Scale (EDSS) score, annualized relapse rate (ARR), and disease-modifying therapy (DMT) non-adherence. Results Among 99 Latino patients, 84 had MS, 11 MOGAD, and 4 NMOSD; 46.5% lived [&ge;]50 miles from clinic. Living [&ge;]50 miles from clinic was associated with higher EDSS scores in unadjusted analyses, but not after covariate adjustment. In multivariable analyses, Medicaid insurance was associated with higher EDSS compared with commercial insurance ({beta}=1.071, p=0.031) and higher ARR ({beta}=0.230, p=0.022). Higher ADI showed a non-significant trend toward higher EDSS ({beta}=0.147 per 1-decile increase, p=0.068). DMT non-adherence was not significantly associated with covariates. Conclusions In this cohort of Latinos with CNS demyelinating diseases, Medicaid insurance was associated with greater disability level and higher relapse activity. These findings suggest that insurance status should be considered when designing strategies to improve access to neuroimmunology care.

Objective Astrocyte activation is increasingly recognized as an important component of multiple sclerosis (MS) pathology. Natalizumab (NTZ), a highly effective therapy for relapsing-remitting MS (RRMS), primarily blocks leukocyte trafficking into the central nervous system. However, its effects on astrocytic metabolism remain unclear. We investigated astrocyte-associated metabolic changes after NTZ treatment using quantitative 1-11C-acetate positron emission tomography (PET). Methods Seven patients with RRMS underwent quantitative 1-11C-acetate PET before and after NTZ treatment. PET-derived k2, an index of oxidative acetate metabolism, was analyzed voxel-wise and within GM and white-matter volumes of interest. Clinical status and brain magnetic resonance imaging (MRI) findings were assessed, and cognitive performance was evaluated using Rao's Brief Repeatable Battery of Neuropsychological Tests. Results After NTZ treatment, k2 decreased in all patients compared with pretreatment levels. Both gray and white matter showed significant reductions, and voxel-based analysis demonstrated widespread decreases across cortical and subcortical regions of the cerebrum and cerebellum, with no regions showing significant posttreatment increases. MRI showed no worsening; Expanded Disability Status Scale scores were stable or improved, and cognitive performance was generally stable, with improvements in selected subtests. Interpretation Quantitative 1-11C-acetate PET demonstrated a whole-brain reduction in astrocyte-associated metabolism after NTZ treatment in RRMS, most prominently in gray matter. NTZ may modulate astrocyte activity, in addition to its established effects on peripheral immune cell trafficking.

CNS oligodendrocytes generate myelin, an RNA-containing proteolipid substance that enhances axonal transmission. In multiple sclerosis (MS), myelin debris is phagocytosed by microglia (MG), and prior studies have detected myelin-derived mRNA in MG nuclei, suggesting a retrograde transport pathway. We report myelin basic protein (MBP) is a nucleic acid-binding and trafficking protein. We found that retro-transport of myelin RNA into the MG nucleus was phagocytosis and importin-dependent. Transcriptomic and proteomic analyses of MG nuclei revealed enrichment of myelin mRNAs and proteins, with MBP singularly detected in soluble and chromatin-associated fractions. MBP bound mRNA with high affinity (Kd {approx} 0.30 nM) and was sufficient to facilitate MG RNA nuclear import in vitro and in vivo. Functionally, MBP mediated the delivery of small interfering RNAs for targeted knockdown of toll-like receptor 4. These findings indicate MBP as an RNA-binding protein capable of MG nuclear import, providing insight into neuroinflammatory pathology of MS.