Inhibitors of Bruton tyrosine kinase (BTK) are actively being pursued as potential disease-modifying therapies for multiple sclerosis (MS), as attested by several completed or ongoing Phase 3 clinical trials. Yet, key aspects of BTK inhibitors biology remain unclear. Here, we assessed the effects of remibrutinib, a BTK inhibitor in Phase 3 MS trials, in preclinical models of MS. Remibrutinib was evaluated in the myelin oligodendrocyte glycoprotein (MOG)35-55 model of experimental autoimmune encephalomyelitis (EAE) and in the lysolecithin model of demyelination. In culture, we assessed its ability to reduce tumor necrosis factor-α (TNF-α) elicited by immune complex-activation of primary human and mouse microglia, mouse macrophages, and human inducible pluripotent stem cell (iPS)-derived microglia. We also examined the impact of remibrutinib on T-bet+ memory B cells, increasingly implicated in neuroinflammation. Remibrutinib attenuated clinical disability of MOG-induced EAE, accompanied by decreased lesional burden in the spinal cord. In lysolecithin-induced demyelination, remibrutinib did not affect oligodendrogenesis in young mice with robust repair capacity, or in middle-aged mice where remyelination is deficient. Across microglia and macrophage cultures of human or murine origin, remibrutinib effectively reduced immune complex-induced TNF-α. Multiplex cytokine analyses showed that this reduction coincided with increased interleukin-10, suggesting a shift toward a more regulatory functional state. In T-bet+ memory B cells, remibrutinib decreased production of pro-inflammatory cytokines in culture and reduced expression of markers associated with T-bet+ memory B cells in mice. While Phase 3 clinical trial results in MS are awaited, the drug favorably affects inflammatory outcomes in MS models.

The germinal center (GC) reaction requires tight regulation of B cell and T follicular helper (Tfh) cell interactions to ensure B cell expansion and antibody affinity maturation, while preventing oncogenesis. However, regulatory mechanisms fine-tuning B-T cell interactions within the GC to prevent aberrant activation and proliferation remain incompletely understood. Here, we identify Siglec-G, the mouse ortholog of human Siglec-10, as an immune checkpoint that restrains the GC by dampening B-T cell interactions. Selective and temporal ablation of Siglec-G on B cells after immunization triggers GC hyperplasia and enhanced plasma cell and antibody output. While Siglec-G is dispensable in B cell receptor (BCR)-mediated processes, it acts as an intrinsic inhibitory receptor of B-T cell interactions in the GC, ultimately limiting Myc and mTORC activation within positively selected GC B cells. Trans interactions of Siglec-G and its glycan ligands on Tfh likely contribute in fine-tuning the strength of bidirectional signaling following contact between GC B cells and Tfh cells. This interaction is further reinforced by glycan remodeling that occurs in the GC, resulting in concurrent decreased in glycan ligands on GC B cells and increased in glycan ligands on Tfh. This augmented binding of Siglec-G/10 on Tfh is mainly due to the upregulation of α2-6 linked sialic acid ligands. Moreover, APEX2-based proximity labeling revealed several candidate Siglec-G/10 binding partners on T cells, including BTLA, CD6, and Slamf6, which are known negative regulators of Tfh cell activation. Taken together, our findings identified that Siglec-G acts as a GC checkpoint receptor, restricting B cell proliferation by tuning T cell help following B-T cell interactions.

BACKGROUND:Despite growing evidence on serum neurofilament light (sNfL) as a biomarker in Multiple Sclerosis (MS), its implementation and performance in clinical practice remain underexplored. This study aimed to evaluate the application of NfL testing comparing two immunoassays-the current gold-standard Simoa by Quanterix and the most recent Elecsys by Roche-in real-world conditions, moving beyond controlled research environments. METHOD:This cross-sectional study recruited 97 patients with Relapsing-Remitting MS (RRMS), collecting baseline and retrospective demographic, clinical and MRI data as part of routine care. Serum NfL levels were measured using Simoa and Elecsys immunoassays and were log10-transformed. Associations between sNfL levels and outcomes were evaluated using generalized linear and binary logistic regression models, with covariate adjustments reflective of clinical conditions. RESULTS:Elecsys sNfL highly correlated with Simoa levels (R2≈0.891) with a strong negative bias of 88.19 %. Despite this, both assays showed that sNfL significantly associated with disease-modifying treatment, time since last relapse, Expanded Disability Status Scale, Symbol Digit Modalities Test, Timed 25-Foot Walk, brain atrophy, GD-enhancing T2 brain lesions, new or enlarging T2 brain lesions and infratentorial T2 brain lesions. Subgroup analyses showed Elecsys and Simoa sNfL models had similar performance metrics compared to clinical-only models, though Elecsys sNfL demonstrated mixed findings with some markers of disease activity (e.g. GD+ lesions) and progression (e.g. T1-hypointense, infratentorial and cervical spinal cord lesion burden). CONCLUSION:Our results support the use of sNfL as a robust and informative biomarker within the real-world complexities of clinical care, with either the Elecsys or Simoa immunoassays offering reliable options.

ABSTRACT Objective The cervical spinal cord (cSC) is highly relevant to clinical dysfunction in multiple sclerosis (MS) but remains understudied using quantitative magnetic resonance imaging (MRI). We assessed magnetization transfer ratio (MTR), a semi‐quantitative MRI measure sensitive to MS‐related tissue microstructural changes, in the cSC and its relationship with clinical outcomes in radiologically isolated syndrome (RIS) and MS. Methods MTR data were acquired from 52 RIS, 201 relapsing–remitting MS (RRMS), 47 primary progressive MS (PPMS), and 43 control (CON) participants across four sites in the Canadian Prospective Cohort Study to Understand Progression in MS (CanProCo) using 3.0 T MRI systems. Mean MTR was compared between groups in whole cSC and sub‐regions between C2‐C4. Multiple linear regression was used to evaluate relationships between MTR and clinical outcomes, including the expanded disability status scale (EDSS), walking speed test (WST), and manual dexterity test (MDT). Results There were consistent group differences in MTR, which were most pronounced between PPMS and CON (−5.8% to −3.7%, p ≤ 0.01). In PPMS, lower MTR was associated with greater disability as measured by EDSS ( β = −0.3 to −0.1, p ≤ 0.03), WST ( β = −0.9 to −0.5, p ≤ 0.04), and MDT ( β = −0.6 and − 0.5, p = 0.04). In RRMS, MTR was associated with only EDSS ( β = −0.1, p ≤ 0.03). Interpretation In this large sample of RIS and MS, cSC MTR was lowest in PPMS, with associations between MTR and clinical outcomes in MS but not RIS. These findings suggest that MTR provides important information about the underlying tissue microstructural integrity of the cSC relevant to clinical disability in established MS.

Real-world studies in the USA report that 41–56

Background: Disease progression is observed across the spectrum of people with multiple sclerosis (MS) and identification of effective treatment strategies to halt progression remains one of the greatest unmet clinical needs. Objectives: The Canadian Prospective Cohort Study to Understand Progression in MS (CanProCo) was designed to evaluate a wide range of factors associated with the onset and rate of clinical disease progression in MS and to describe the interplay between these factors. Design: A prospective cohort study. Methods: CanProCo is a national, prospective, observational cohort study that has recruited 944 individuals from 5 large academic MS centers in Canada. Participants include people with radiologically isolated syndrome (RIS), early relapsing-remitting and primary progressive MS (RRMS, PPMS), and healthy controls (HCs). Annually, participants complete self-reported questionnaires, undergo clinical evaluation and, if clinically indicated, magnetic resonance images (MRIs) of the brain and cervical spinal cord; in a subset of participants ( n = 399), blood, and research MRIs of the brain and cervical spinal cord are collected. Linkages to health administrative databases are available at three sites. Results: Overall, 944 participants were recruited (53 HCs, 63 RIS, 751 RRMS, 77 PPMS). RIS and MS participants had a mean age of 39.0 years and 70.5% female. The mean time since diagnosis was 2.7 years. There were differences observed in the Expanded Disability Status Scale score and components of the MS performance test (walking speed test, manual dexterity test, processing speed test, and low-contrast visual acuity) between RIS and MS subtypes. Questionnaires revealed more symptoms of depression and anxiety and impaired physical and mental quality of life in people with RIS/MS versus HCs and differences across RIS/MS subtypes. Conclusion: Physical and mental neurological disability is prevalent even in the earliest stages of MS. Transdisciplinary approaches such as those used in CanProCo are needed to better characterize clinical progression in MS. Additional CanProCo results, including MRI, biological, and pharmaco-economic data will be forthcoming. Going forward, CanProCo’s data sharing and collaborative vision will facilitate numerous global collaborations, which will inform the development and implementation of effective interventions for people with MS around the world. Keywords biomarkers , cohort , epidemiology , immunophenotyping , MRI , multiple sclerosis , progression

Intermediate-length repeat expansions in ATAXIN-2 (ATXN2) are the strongest genetic risk factor for amyotrophic lateral sclerosis (ALS). At the molecular level, ATXN2 intermediate expansions enhance TDP-43 toxicity and pathology. However, whether this triggers ALS pathogenesis at the cellular and functional level remains unknown. Here, we combine patient-derived and mouse models to dissect the effects of ATXN2 intermediate expansions in an ALS background. iPSC-derived motor neurons from ATXN2-ALS patients show altered stress granules, neurite damage and abnormal electrophysiological properties compared to healthy control and other familial ALS mutations. In TDP-43Tg-ALS mice, ATXN2-Q33 causes reduced motor function, NMJ alterations, neuron degeneration and altered in vitro stress granule dynamics. Furthermore, gene expression changes related to mitochondrial function and inflammatory response are detected and confirmed at the cellular level in mice and human neuron and organoid models. Together, these results define pathogenic defects underlying ATXN2-ALS and provide a framework for future research into ATXN2-dependent pathogenesis and therapy.

Demyelinating disorders of the central nervous system (CNS) occur when myelin and oligodendrocytes are damaged or lost. Remyelination and regeneration of oligodendrocytes can be achieved from endogenous oligodendrocyte precursor cells (OPCs) that reside in the adult CNS tissue. Using a cuprizone mouse model of demyelination, we show that infusion of fractalkine (CX3CL1) into the demyelinated murine brain increases de novo oligodendrocyte formation and enhances remyelination in the corpus callosum and cortical gray matter. This is achieved by increased OPC proliferation in the cortical gray matter as well as OPC differentiation and attenuation of microglia/macrophage activation both in corpus callosum and cortical gray matter. Finally, we show that activated OPCs and microglia/macrophages express fractalkine receptor CX3CR1 in vivo, and that in OPC-microglia co-cultures fractalkine increases in vitro oligodendrocyte differentiation by modulating both OPC and microglia biology. Our results demonstrate a novel pro-regenerative role of fractalkine in a demyelinating mouse model.

Biodegradable poly(lactic-co-glycolic acid)(PLGA) nanoparticles have been used extensively in delivering drugs to target tissues due to their excellent biocompatibility. Evidence suggests that PLGA-conjugated drugs/agents can attenuate pathology in cellular/animal models of Alzheimer's disease (AD), which is initiated by increased level/aggregation of amyloid β (Aβ) peptide generated from amyloid precursor protein (APP). The beneficial effects were attributed to conjugated-drugs rather than to PLGA nanoparticles. Interestingly, we recently reported that PLGA without any drug/agent (native PLGA) can suppress Aβ aggregation/toxicity. However, very little is known about the internalization, subcellular localization or effects of PLGA in neurons. In this study, using primary mouse cortical neurons, we first showed that native PLGA is internalized by an energy-mediated clathrin-dependent/-independent pathway and is localized in endosomal-lysosomal-autophagic vesicles. By attenuating internalization, PLGA can protect neurons against Aβ-mediated toxicity. Additionally, PLGA treatment altered expression profiles of certain AD-associated genes and decreased the levels of APP, its cleaved products α-/β-CTFs and Aβ peptides in mouse as well as iPSC-derived neurons from control and AD patients. Collectively, these results suggest that native PLGA not only protects neurons against Aβ-induced toxicity but also influences the expression of AD-related genes/proteins - highlighting PLGA's implication in normal and AD-related pathology.

Multiple sclerosis (MS) and its animal models are characterized by cellular inflammation within the central nervous system (CNS). The sources and consequences of this inflammation are currently not completely understood. Critical signs and mediators of CNS inflammation are reactive oxygen species (ROS) that promote inflammation. ROS originate from a variety of redox-reactive enzymes, one class of which catalyses oxidative protein folding within the endoplasmic reticulum (ER). Here, the unfolded protein response and other signalling mechanisms maintain a balance between ROS producers such as ER oxidoreductin 1α (Ero1α) and antioxidants such as glutathione peroxidase 8 (GPx8). The role of ROS production within the ER has so far not been examined in the context of MS. In this manuscript, we examined how components of the ER redox network change upon MS and experimental autoimmune encephalomyelitis (EAE). We found that unlike GPx8, Ero1α increases within both MS and EAE astrocytes, in parallel with an imbalance of other oxidases such of GPx7, and that no change was observed within neurons. This imbalance of ER redox enzymes can reduce the lifespan of astrocytes, while neurons are not affected. Therefore, Ero1α induction makes astrocytes vulnerable to oxidative stress in the MS and EAE pathologies.

Abstract Background Gangliosides are glycosphingolipids highly enriched in the brain, with important roles in cell signaling, cell-to-cell communication, and immunomodulation. Genetic defects in the ganglioside biosynthetic pathway result in severe neurodegenerative diseases, while a partial decrease in the levels of specific gangliosides was reported in Parkinson’s disease and Huntington’s disease. In models of both diseases and other conditions, administration of GM1—one of the most abundant gangliosides in the brain—provides neuroprotection. Most studies have focused on the direct neuroprotective effects of gangliosides on neurons, but their role in other brain cells, in particular microglia, is not known. In this study we investigated the effects of exogenous ganglioside administration and modulation of endogenous ganglioside levels on the response of microglia to inflammatory stimuli, which often contributes to initiation or exacerbation of neurodegeneration. Methods In vitro studies were performed using BV2 cells, mouse, rat, and human primary microglia cultures. Modulation of microglial ganglioside levels was achieved by administration of exogenous gangliosides, or by treatment with GENZ-123346 and L–t-PDMP, an inhibitor and an activator of glycolipid biosynthesis, respectively. Response of microglia to inflammatory stimuli (LPS, IL-1β, phagocytosis of latex beads) was measured by analysis of gene expression and/or secretion of pro-inflammatory cytokines. The effects of GM1 administration on microglia activation were also assessed in vivo in C57Bl/6 mice, following intraperitoneal injection of LPS. Results GM1 decreased inflammatory microglia responses in vitro and in vivo, even when administered after microglia activation. These anti-inflammatory effects depended on the presence of the sialic acid residue in the GM1 glycan headgroup and the presence of a lipid tail. Other gangliosides shared similar anti-inflammatory effects in in vitro models, including GD3, GD1a, GD1b, and GT1b. Conversely, GM3 and GQ1b displayed pro-inflammatory activity. The anti-inflammatory effects of GM1 and other gangliosides were partially reproduced by increasing endogenous ganglioside levels with L–t-PDMP, whereas inhibition of glycolipid biosynthesis exacerbated microglial activation in response to LPS stimulation. Conclusions Our data suggest that gangliosides are important modulators of microglia inflammatory responses and reveal that administration of GM1 and other complex gangliosides exerts anti-inflammatory effects on microglia that could be exploited therapeutically.

Multiple sclerosis (MS) and its animal models are characterized by cellular inflammation within the central nervous system (CNS). The sources and consequences of this inflammation are currently not completely understood. Critical signs and mediators of CNS inflammation are reactive oxygen species (ROS) that promote inflammation. ROS originate from a variety of redox-reactive enzymes, one class of which catalyses oxidative protein folding within the endoplasmic reticulum (ER). Here, the unfolded protein response and other signalling mechanisms maintain a balance between ROS producers such as ER oxidoreductin 1 alpha (Ero1 alpha) and antioxidants such as glutathione peroxidase 8 (GPx8). The role of ROS production within the ER has so far not been examined in the context of MS. In this manuscript, we examined how components of the ER redox network change upon MS and experimental autoimmune encephalomyelitis (EAE). We found that unlike GPx8, Ero1 alpha increases within both MS and EAE astrocytes, in parallel with an imbalance of other oxidases such of GPx7, and that no change was observed within neurons. This imbalance of ER redox enzymes can reduce the lifespan of astrocytes, while neurons are not affected. Therefore, Ero1 alpha induction makes astrocytes vulnerable to oxidative stress in the MS and EAE pathologies.

Alzheimer's disease (AD) is believed to be triggered by increased levels/aggregation of β-amyloid (Aβ) peptides. At present, there is no effective disease-modifying treatment for AD. Here, we evaluated the therapeutic potential of FDA-approved native poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles on Aβ aggregation and in cellular/animal models of AD. Our results showed that native PLGA can not only suppress the spontaneous aggregation but can also trigger disassembly of preformed Aβ aggregates. Spectroscopic studies, molecular dynamics simulations and biochemical analyses revealed that PLGA, by interacting with the hydrophobic domain of Aβ1-42, prevents a conformational shift towards the β-sheet structure, thus precluding the formation and/or triggering disassembly of Aβ aggregates. PLGA-treated Aβ samples can enhance neuronal viability by reducing phosphorylation of tau protein and its associated signaling mechanisms. Administration of PLGA can interact with Aβ aggregates and attenuate memory deficits as well as Aβ levels/deposits in the 5xFAD mouse model of AD. PLGA can also protect iPSC-derived neurons from AD patients against Aβ toxicity by decreasing tau phosphorylation. These findings provide unambiguous evidence that native PLGA, by targeting different facets of the Aβ axis, can have beneficial effects in mouse neurons/animal models as well as on iPSC-derived AD neurons - thus signifying its unique therapeutic potential in the treatment of AD pathology.

Background Increasing evidence suggests that various inflammatory, immunological and metabolic pathways are altered in the clinically isolated syndrome (CIS) of multiple sclerosis (MS). Moreover, recent diagnostic criteria have made possible the very early diagnosis of MS. We evaluated multiple fluid biomarkers in people with early MS and CIS. Methods We measured blood levels of cytokines, matrix metalloproteinases (MMPs), serum metabolomics and immune cell immunophenotyping in participants in the Trial of Minocycline in a Clinically Isolated Syndrome of Multiple Sclerosis. Results When compared with healthy controls, people with early MS/CIS had higher levels of eotaxin, MCP-3, IL-1 receptor antagonist, IL-1β, IL-9 and IP-10, as well as MMPs 1, 8 and 9. In metabolomics analysis, the alanine, aspartate and glutamate metabolism and the synthesis and degradation of ketone bodies pathways were altered compared to healthy controls. There were no differences in lymphocyte subpopulation numbers. Out of all these biomarkers, only MMP-1 was able to differentiate between early MS and CIS, and was found to correlate with lesion volume and gadolinium enhancing lesions on MRI. Conclusion The immunological and metabolic profile of CIS and early MS is remarkably similar, supporting that these are a continuum of a common underlying pathophysiological process.

BACKGROUND:Relapsing-remitting multiple sclerosis (RRMS) is an autoimmune disorder of the central nervous system. Dimethyl Fumarate is a disease-modifying medication used to treat RRMS patients that can induce lymphopenia. We aimed to immunophenotype peripheral blood mononuclear cells (PBMC) in RRMS patients cross-sectionally and examine the characteristics and modifications of lymphopenia over time.METHODS:Characterization of PBMC was done by multiparametric flow cytometry. Patients had been on treatment for up to 4 years and were grouped into lymphopenic (DMF-L) and non-lymphopenic (DMF-N) patients.RESULTS:Lymphopenia affected the cell population changes over time, with other patient characteristics (gender, age, and previous treatment status) also having significant effects. In both lymphopenic and non-lymphopenic patients, PBMC percentages were reduced over time. While overall T and B cells frequencies were not affected, males, older patients and untreated patients had significant changes in B cell subpopulations over time. CD4+ to CD8+T cell ratio increased significantly in lymphopenic patients over time. CD4-CD8-T cell population was similarly reduced in both lymphopenic and non-lymphopenic patients, over time. While the monocyte and NK overall populations were not changed, non-classical monocyte subpopulation decreased over time in lymphopenic patients. We also found CD56-CD16+ and CD56-CD16- NK cells frequencies changed over time in lymphopenic patients. Immune populations showed correlations with clinical outcomes measured by EDSS and relapse rate. Analysis of the overall immunophenotype showed that, while groups divided by other patient characteristics showed differences, the lymphopenia status overrode these differences, resulting in similar immunophenotype within DMF-L.CONCLUSIONS:Our data provide evidence that under the same therapy, lymphopenia affects how the immunophenotype changes over time and can override the differences associated with other patient characteristics and possibly mask other significant changes in the immune profile of patients.

BACKGROUND:Neuroactive steroids (NASs) exert multiple biological effects on development and inflammation. The effects of NASs on disease progression in multiple sclerosis (MS) are uncertain, prompting analyses of NAS profiles during the transition from clinically isolated syndrome (CIS) to relapsing-remitting (RR) MS.METHODS:Subjects with CIS or RRMS and healthy controls (HCs) were recruited; demographic and clinical data as well as disability scores measured by the Expanded Disability Status Scale (EDSS) were recorded. Matched plasma NAS and amino acid (AA) concentrations were measured.RESULTS:HC (n = 17), CIS (n = 31), and RRMS (n = 33) groups showed similar ages and sex distribution although disability scores were higher in the RRMS group. The conversion rate of CIS to RRMS group was 51.6% (n = 16) during a mean follow-up period of 1.85 years. The RRMS group showed significantly higher mean allopregnanolone, aspartate, and taurine concentrations with lower epiallopregnanolone concentrations than CIS patients, and higher L-serine-O-phosphate and lower alanine, arginine, and glutamine concentrations than the HC group. Among CIS and RRMS groups, multivariate hierarchical regressions revealed that higher concentrations of plasma tetrahydrodeoxycorticosterone (THDOC) may predict disability worsening.CONCLUSIONS:RRMS and CIS patients exhibited differing concentrations of both NASs and AAs in plasma while both THDOC and pregnanolone might serve as biomarkers of disability worsening.