Interleukin-3 coordinates glial-peripheral immune crosstalk to incite multiple sclerosis.

Glial cells and central nervous system (CNS)-infiltrating leukocytes contribute to multiple sclerosis (MS). However, the networks that govern crosstalk among these ontologically distinct populations remain unclear. Here, we show that, in mice and humans, CNS-resident astrocytes and infiltrating CD44hiCD4+ T cells generated interleukin-3 (IL-3), while microglia and recruited myeloid cells expressed interleukin-3 receptor-ɑ (IL-3Rɑ). Astrocytic and T cell IL-3 elicited an immune migratory and chemotactic program by IL-3Rɑ+ myeloid cells that enhanced CNS immune cell infiltration, exacerbating MS and its preclinical model. Multiregional snRNA-seq of human CNS tissue revealed the appearance of IL3RA-expressing myeloid cells with chemotactic programming in MS plaques. IL3RA expression by plaque myeloid cells and IL-3 amount in the cerebrospinal fluid predicted myeloid and T cell abundance in the CNS and correlated with MS severity. Our findings establish IL-3:IL-3RA as a glial-peripheral immune network that prompts immune cell recruitment to the CNS and worsens MS.

Cell-binding IgM in CSF is distinctive of multiple sclerosis and targets the iron transporter SCARA5.

Intrathecal IgM production in multiple sclerosis is associated with a worse disease course. To investigate pathogenic relevance of autoreactive IgM in multiple sclerosis, CSF from two independent cohorts, including multiple sclerosis patients and controls, were screened for antibody binding to induced pluripotent stem cell-derived neurons and astrocytes, and a panel of CNS-related cell lines. IgM binding to a primitive neuro-ectodermal tumour cell line discriminated 10% of multiple sclerosis donors from controls. Transcriptomes of single IgM producing CSF B cells from patients with cell-binding IgM were sequenced and used to produce recombinant monoclonal antibodies for characterization and antigen identification. We produced five cell-binding recombinant IgM antibodies, of which one, cloned from an HLA-DR + plasma-like B cell, mediated antigen-dependent complement activation. Immunoprecipitation and mass spectrometry, and biochemical and transcriptome analysis of the target cells identified the iron transport scavenger protein SCARA5 as the antigen target of this antibody. Intrathecal injection of a SCARA5 antibody led to an increased T cell infiltration in an experimental autoimmune encephalomyelitis (EAE) model. CSF IgM might contribute to CNS inflammation in multiple sclerosis by binding to cell surface antigens like SCARA5 and activating complement, or by facilitating immune cell migration into the brain.

High-dimensional immune profiling identifies a biomarker to monitor dimethyl fumarate response in multiple sclerosis.

Dimethyl fumarate (DMF) is an immunomodulatory treatment for multiple sclerosis (MS). Despite its wide clinical use, the mechanisms underlying clinical response are not understood. This study aimed to reveal immune markers of therapeutic response to DMF treatment in MS. For this purpose, we prospectively collected peripheral blood mononuclear cells (PBMCs) from a highly characterized cohort of 44 individuals with MS before and at 12 and 48 wk of DMF treatment. Single cells were profiled using high-dimensional mass cytometry. To capture the heterogeneity of different immune subsets, we adopted a bioinformatic multipanel approach that allowed cell population-cluster assignment of more than 50 different parameters, including lineage and activation markers as well as chemokine receptors and cytokines. Data were further analyzed in a semiunbiased fashion implementing a supervised representation learning approach to capture subtle longitudinal immune changes characteristic for therapy response. With this approach, we identified a population of memory T helper cells expressing high levels of neuroinflammatory cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF], interferon γ [IFNγ]) as well as CXCR3, whose abundance correlated with treatment response. Using spectral flow cytometry, we confirmed these findings in a second cohort of patients. Serum neurofilament light-chain levels confirmed the correlation of this immune cell signature with axonal damage. The identified cell population is expanded in peripheral blood under natalizumab treatment, substantiating a specific role in treatment response. We propose that depletion of GM-CSF-, IFNγ-, and CXCR3-expressing T helper cells is the main mechanism of action of DMF and allows monitoring of treatment response.

Emulating randomised clinical trials in relapsing-remitting multiple sclerosis with non-randomised real-world evidence: an application using data from the MSBase Registry.

BACKGROUND: To mimic as closely as possible a randomised controlled trial (RCT) and calibrate the real-world evidence (RWE) studies against a known treatment effect would be helpful to understand if RWE can support causal conclusions in selected circumstances. The aim was to emulate the TRANSFORMS trial comparing Fingolimod (FTY) versus intramuscular interferon ß-1a (IFN) using observational data. METHODS: We extracted from the MSBase registry all the patients with relapsing-remitting multiple sclerosis (RRMS) collected in the period 2011-2021 who received IFN or FTY (0.5 mg) and with the same inclusion and exclusion criteria of the TRANSFORMS RCT. The primary endpoint was the annualised relapse rate (ARR) over 12 months. Patients were 1:1 propensity-score (PS) matched. Relapse-rate ratio (RR) was calculated by mean of a negative binomial regression. RESULTS: A total of 4376 patients with RRMS (1140 in IFN and 3236 in FTY) were selected. After PS, 856 patients in each group were matched. The ARR was 0.45 in IFN and 0.25 in FTY with a significant difference between the two groups (RR: 0.55, 95% CI: 0.45 to 0.68; p<0.001). The result of the emulation was very similar and fell within the 95% CI of that observed in the RCT (RR: 0.49, 95% CI: 0.37 to 0.64; p<0.001) with a standardised difference of 0.66 (p=0.51). CONCLUSIONS: By applying the same inclusion and exclusion criteria used in the RCT and employing appropriate methodology, we successfully replicated the RCT results with only minor discrepancies. Also, even if the confounding bias cannot be fully eliminated, conducting a rigorous target trial emulation could still yield valuable insights for comparative effectiveness research.

Serum biomarker levels predict disability progression in patients with primary progressive multiple sclerosis.

BACKGROUND: We aimed to investigate the potential of serum biomarker levels to predict disability progression in a multicentric real-world cohort of patients with primary progressive multiple sclerosis (PPMS). METHODS: A total of 141 patients with PPMS from 18 European MS centres were included. Disability progression was investigated using change in Expanded Disability Status Scale (EDSS) score over three time intervals: baseline to 2 years, 6 years and to the last follow-up. Serum levels of neurofilament light chain (sNfL), glial fibrillar acidic protein (sGFAP) and chitinase 3-like 1 (sCHI3L1) were measured using single-molecule array assays at baseline. Correlations between biomarker levels, and between biomarkers and age were quantified using Spearman's r. Univariable and multivariable linear models were performed to assess associations between biomarker levels and EDSS change over the different time periods. RESULTS: Median (IQR) age of patients was 52.9 (46.4-58.5) years, and 58 (41.1%) were men. Median follow-up time was 9.1 (7.0-12.6) years. Only 8 (5.7%) patients received treatment during follow-up. sNfL and sGFAP levels were moderately correlated (r=0.43) and both weakly correlated with sCHI3L1 levels (r=0.19 and r=0.17, respectively). In multivariable analyses, levels of the three biomarkers were associated with EDSS changes across all time periods. However, when analysis was restricted to non-inflammatory patients according to clinical and radiological parameters (n=64), only sCHI3L1 levels remained associated with future EDSS change. CONCLUSIONS: Levels of sNfL, sGFAP and sCHI3L1 are prognostic biomarkers associated with disability progression in patients with PPMS, being CHI3L1 findings less dependent on the inflammatory component associated with disease progression.

NfL reliability across laboratories, stage-dependent diagnostic performance and matrix comparability in genetic FTD: a large GENFI study.

BACKGROUND: Blood neurofilament light chain (NfL) is increasingly considered as a key trial biomarker in genetic frontotemporal dementia (gFTD). We aimed to facilitate the use of NfL in gFTD multicentre trials by testing its (1) reliability across labs; (2) reliability to stratify gFTD disease stages; (3) comparability between blood matrices and (4) stability across recruiting sites. METHODS: Comparative analysis of blood NfL levels in a large gFTD cohort (GENFI) for (1)-(4), with n=344 samples (n=148 presymptomatic, n=11 converter, n=46 symptomatic subjects, with mutations in C9orf72, GRN or MAPT; and n=139 within-family controls), each measured in three different international labs by Simoa HD-1 analyzer. RESULTS: NfL revealed an excellent consistency (intraclass correlation coefficient (ICC) 0.964) and high reliability across the three labs (maximal bias (pg/mL) in Bland-Altman analysis: 1.12±1.20). High concordance of NfL across laboratories was moreover reflected by high areas under the curve for discriminating conversion stage against the (non-converting) presymptomatic stage across all three labs. Serum and plasma NfL were largely comparable (ICC 0.967). The robustness of NfL across 13 recruiting sites was demonstrated by a linear mixed effect model. CONCLUSIONS: Our results underline the suitability of blood NfL in gFTD multicentre trials, including cross-lab reliable stratification of the highly trial-relevant conversion stage, matrix comparability and cross-site robustness.

Routine CSF parameters as predictors of disease course in multiple sclerosis: an MSBase cohort study.

BACKGROUND: It remains unclear whether routine cerebrospinal fluid (CSF) parameters can serve as predictors of multiple sclerosis (MS) disease course. METHODS: This large-scale cohort study included persons with MS with CSF data documented in the MSBase registry. CSF parameters to predict time to reach confirmed Expanded Disability Status Scale (EDSS) scores 4, 6 and 7 and annualised relapse rate in the first 2 years after diagnosis (ARR2) were assessed using (cox) regression analysis. RESULTS: In total, 11 245 participants were included of which 93.7% (n=10 533) were persons with relapsing-remitting MS (RRMS). In RRMS, the presence of CSF oligoclonal bands (OCBs) was associated with shorter time to disability milestones EDSS 4 (adjusted HR=1.272 (95% CI, 1.089 to 1.485), p=0.002), EDSS 6 (HR=1.314 (95% CI, 1.062 to 1.626), p=0.012) and EDSS 7 (HR=1.686 (95% CI, 1.111 to 2.558), p=0.014). On the other hand, the presence of CSF pleocytosis (≥5 cells/µL) increased time to moderate disability (EDSS 4) in RRMS (HR=0.774 (95% CI, 0.632 to 0.948), p=0.013). None of the CSF variables were associated with time to disability milestones in persons with primary progressive MS (PPMS). The presence of CSF pleocytosis increased ARR2 in RRMS (adjusted R2=0.036, p=0.015). CONCLUSIONS: In RRMS, the presence of CSF OCBs predicts shorter time to disability milestones, whereas CSF pleocytosis could be protective. This could however not be found in PPMS. CSF pleocytosis is associated with short-term inflammatory disease activity in RRMS. CSF analysis provides prognostic information which could aid in clinical and therapeutic decision-making.

Use of pragmatic randomized trials in multiple sclerosis: A systematic overview.

BACKGROUND: Pragmatic trials are increasingly recognized for providing real-world evidence on treatment choices. OBJECTIVE: The objective of this study is to investigate the use and characteristics of pragmatic trials in multiple sclerosis (MS). METHODS: Systematic literature search and analysis of pragmatic trials on any intervention published up to 2022. The assessment of pragmatism with PRECIS-2 (PRagmatic Explanatory Continuum Indicator Summary-2) is performed. RESULTS: We identified 48 pragmatic trials published 1967-2022 that included a median of 82 participants (interquartile range (IQR) = 42-160) to assess typically supportive care interventions (n = 41; 85%). Only seven trials assessed drugs (15%). Only three trials (6%) included >500 participants. Trials were mostly from the United Kingdom (n = 18; 38%), Italy (n = 6; 13%), the United States and Denmark (each n = 5; 10%). Primary outcomes were diverse, for example, quality-of-life, physical functioning, or disease activity. Only 1 trial (2%) used routinely collected data for outcome ascertainment. No trial was very pragmatic in all design aspects, but 14 trials (29%) were widely pragmatic (i.e. PRECIS-2 score ⩾ 4/5 in all domains). CONCLUSION: Only few and mostly small pragmatic trials exist in MS which rarely assess drugs. Despite the widely available routine data infrastructures, very few trials utilize them. There is an urgent need to leverage the potential of this pioneering study design to provide useful randomized real-world evidence.

Efficacy and safety results after >3.5 years of treatment with the Bruton's tyrosine kinase inhibitor evobrutinib in relapsing multiple sclerosis: Long-term follow-up of a Phase II randomised clinical trial with a cerebrospinal fluid sub-study.

BACKGROUND: Evobrutinib - an oral, central nervous system (CNS)-penetrant, and highly selective Bruton's tyrosine kinase inhibitor - has shown efficacy in a 48-week, double-blind, Phase II trial in patients with relapsing MS. OBJECTIVE: Report results of the Phase II open-label extension (OLE; up to week 192 from randomisation) and a cerebrospinal fluid (CSF) sub-study. METHODS: In the 48-week double-blind period (DBP), patients received evobrutinib 25 mg once-daily, 75 mg once-daily, 75 mg twice-daily or placebo (switched to evobrutinib 25 mg once-daily after week 24). Patients could then enter the OLE, receiving evobrutinib 75 mg once-daily (mean (± standard deviation (SD)) duration = 50.6 weeks (±6.0)) before switching to 75 mg twice-daily. RESULTS: Of 164 evobrutinib-treated patients who entered the OLE, 128 (78.0%) completed ⩾192 weeks of treatment. Patients receiving DBP evobrutinib 75 mg twice-daily: annualised relapse rate at week 48 (0.11 (95% confidence interval (CI) = 0.04-0.25)) was maintained with the OLE twice-daily dose up to week 192 (0.11 (0.05-0.22)); Expanded Disability Status Scale score remained stable; serum neurofilament light chain fell to levels like a non-MS population (Z-scores); T1 gadolinium-enhancing lesion numbers remained low. No new safety signals were identified. In the OLE, evobrutinib was detected in the CSF of all sub-study patients. CONCLUSION: Long-term evobrutinib treatment was well tolerated and associated with a sustained low level of disease activity. Evobrutinib was present in CSF at concentrations similar to plasma.

Effects of obstructive sleep apnea treatment on neurodegenerative biomarker neurofilament light chain and cognitive performance.

Obstructive sleep apnea is associated with cognitive impairment and increased risk for neurodegenerative diseases. Obstructive sleep apnea treatment with positive airway pressure therapy helps to improve cognitive symptoms and reduces long-term dementia risk. To test whether these treatment effects are due to a reduction in neuronal damage, we examined longitudinal changes in the neurodegenerative serum neurofilament light chain and cognitive performance of patients with obstructive sleep apnea. In this study, 17 patients with obstructive sleep apnea completed baseline and follow-up (9 month after starting PAP treatment) investigation of sleep, daytime symptoms, cognitive testing and serum neurofilament light chain measurements. Depending on treatment adherence and efficacy, participants were assigned either to the effective treatment (n = 10) or non-effective treatment group (n = 7). As results at baseline lower mean oxygen saturation during sleep was associated with higher serum neurofilament light chain. Patients in the non-effective treatment group showed a significant increase of age-adjusted percentile of serum neurofilament light chain levels at follow-up, whereas serum neurofilament light chain values remained constant in the effective treatment group. At a functional level, effective treatment leads to an improvement in processing speed, which was not the case in the non-effective treatment group. Longitudinal changes of age-adjusted serum neurofilament light chain levels were associated with changes in cognitive performance. To conclude, this longitudinal observational study showed that effective obstructive sleep apnea treatment positively affects the amount of neuronal damage as well as working memory performance. As cognitive symptoms might not only be attributed to obstructive sleep apnea-related sleep deficiency, but also neurodegeneration, our results underline the importance of treatment adherence and efficacy for the prevention of neuronal damage and cognitive consequences.

Development and multi-center validation of a fully automated digital immunoassay for neurofilament light chain: toward a clinical blood test for neuronal injury.

OBJECTIVES: Neurofilament light chain (NfL) has emerged as a promising biomarker for detecting and monitoring axonal injury. Until recently, NfL could only be reliably measured in cerebrospinal fluid, but digital single molecule array (Simoa) technology has enabled its precise measurement in blood samples where it is typically 50-100 times less abundant. We report development and multi-center validation of a novel fully automated digital immunoassay for NfL in serum for informing axonal injury status. METHODS: A 45-min immunoassay for serum NfL was developed for use on an automated digital analyzer based on Simoa technology. The analytical performance (sensitivity, precision, reproducibility, linearity, sample type) was characterized and then cross validated across 17 laboratories in 10 countries. Analytical performance for clinical NfL measurement was examined in individual patients with relapsing remitting multiple sclerosis (RRMS) after 3 months of disease modifying treatment (DMT) with fingolimod. RESULTS: The assay exhibited a lower limit of detection (LLoD) of 0.05 ng/L, a lower limit of quantification (LLoQ) of 0.8 ng/L, and between-laboratory imprecision <10 % across 17 validation sites. All tested samples had measurable NfL concentrations well above the LLoQ. In matched pre-post treatment samples, decreases in NfL were observed in 26/29 RRMS patients three months after DMT start, with significant decreases detected in a majority of patients. CONCLUSIONS: The sensitivity characteristics and reproducible performance across laboratories combined with full automation make this assay suitable for clinical use for NfL assessment, monitoring in individual patients, and cross-comparisons of results across multiple sites.

The multifaceted role of neurofilament light chain protein in non-primary neurological diseases.

The advancing validation and exploitation of CSF and blood neurofilament light chain protein as a biomarker of neuroaxonal damage has deeply changed the current diagnostic and prognostic approach to neurological diseases. Further, recent studies have provided evidence of potential new applications of this biomarker also in non-primary neurological diseases. In the present review we summarize the state of the art, future perspectives, but also limitations, of neurofilament light chain protein as a CSF and blood biomarker in several medical fields, including intensive care medicine, surgery, internal medicine and psychiatry. In particular, neurofilament light chain protein is associated with the degree of neurological impairment and outcome in patients admitted to intensive care units or in the perioperative phase and it seems to be highly interconnected with cardiovascular risk factors. Beyond that, interesting diagnostic and prognostic insights have been provided by the investigation of neurofilament light chain protein in psychiatric disorders as well as in the current coronavirus disease-19 pandemic and in normal ageing. Altogether, current data outline a multifaceted applicability of CSF and blood neurofilament light chain protein ranging from the critical clinical setting to the development of precision medicine models suggesting a strict interplay between the nervous system pathophysiology and the health-illness continuum.

The risk of secondary progressive multiple sclerosis is geographically determined but modifiable.

Geographical variations in the incidence and prevalence of multiple sclerosis have been reported globally. Latitude as a surrogate for exposure to ultraviolet radiation but also other lifestyle and environmental factors are regarded as drivers of this variation. No previous studies evaluated geographical variation in the risk of secondary progressive multiple sclerosis, an advanced form of multiple sclerosis that is characterized by steady accrual of irreversible disability. We evaluated differences in the risk of secondary progressive multiple sclerosis in relation to latitude and country of residence, modified by high-to-moderate efficacy immunotherapy in a geographically diverse cohort of patients with relapsing-remitting multiple sclerosis. The study included relapsing-remitting multiple sclerosis patients from the global MSBase registry with at least one recorded assessment of disability. Secondary progressive multiple sclerosis was identified as per clinician diagnosis. Sensitivity analyses used the operationalized definition of secondary progressive multiple sclerosis and the Swedish decision tree algorithm. A proportional hazards model was used to estimate the cumulative risk of secondary progressive multiple sclerosis by country of residence (latitude), adjusted for sex, age at disease onset, time from onset to relapsing-remitting phase, disability (Multiple Sclerosis Severity Score) and relapse activity at study inclusion, national multiple sclerosis prevalence, government health expenditure, and proportion of time treated with high-to-moderate efficacy disease-modifying therapy. Geographical variation in time from relapsing-remitting phase to secondary progressive phase of multiple sclerosis was modelled through a proportional hazards model with spatially correlated frailties. We included 51 126 patients (72% female) from 27 countries. The median survival time from relapsing-remitting phase to secondary progressive multiple sclerosis among all patients was 39 (95% confidence interval: 37 to 43) years. Higher latitude [median hazard ratio = 1.21, 95% credible interval (1.16, 1.26)], higher national multiple sclerosis prevalence [1.07 (1.03, 1.11)], male sex [1.30 (1.22, 1.39)], older age at onset [1.35 (1.30, 1.39)], higher disability [2.40 (2.34, 2.47)] and frequent relapses [1.18 (1.15, 1.21)] at inclusion were associated with increased hazard of secondary progressive multiple sclerosis. Higher proportion of time on high-to-moderate efficacy therapy substantially reduced the hazard of secondary progressive multiple sclerosis [0.76 (0.73, 0.79)] and reduced the effect of latitude [interaction: 0.95 (0.92, 0.99)]. At the country-level, patients in Oman, Tunisia, Iran and Canada had higher risks of secondary progressive multiple sclerosis relative to the other studied regions. Higher latitude of residence is associated with a higher probability of developing secondary progressive multiple sclerosis. High-to-moderate efficacy immunotherapy can mitigate some of this geographically co-determined risk.

N-acetylglucosamine inhibits inflammation and neurodegeneration markers in multiple sclerosis: a mechanistic trial.

BACKGROUND: In the demyelinating disease multiple sclerosis (MS), chronic-active brain inflammation, remyelination failure and neurodegeneration remain major issues despite immunotherapy. While B cell depletion and blockade/sequestration of T and B cells potently reduces episodic relapses, they act peripherally to allow persistence of chronic-active brain inflammation and progressive neurological dysfunction. N-acetyglucosamine (GlcNAc) is a triple modulator of inflammation, myelination and neurodegeneration. GlcNAc promotes biosynthesis of Asn (N)-linked-glycans, which interact with galectins to co-regulate the clustering/signaling/endocytosis of multiple glycoproteins simultaneously. In mice, GlcNAc crosses the blood brain barrier to raise N-glycan branching, suppress inflammatory demyelination by T and B cells and trigger stem/progenitor cell mediated myelin repair. MS clinical severity, demyelination lesion size and neurodegeneration inversely associate with a marker of endogenous GlcNAc, while in healthy humans, age-associated increases in endogenous GlcNAc promote T cell senescence. OBJECTIVES AND METHODS: An open label dose-escalation mechanistic trial of oral GlcNAc at 6 g (n = 18) and 12 g (n = 16) for 4 weeks was performed in MS patients on glatiramer acetate and not in relapse from March 2016 to December 2019 to assess changes in serum GlcNAc, lymphocyte N-glycosylation and inflammatory markers. Post-hoc analysis examined changes in serum neurofilament light chain (sNfL) as well as neurological disability via the Expanded Disability Status Scale (EDSS). RESULTS: Prior to GlcNAc therapy, high serum levels of the inflammatory cytokines IFNγ, IL-17 and IL-6 associated with reduced baseline levels of a marker of endogenous serum GlcNAc. Oral GlcNAc therapy was safe, raised serum levels and modulated N-glycan branching in lymphocytes. Glatiramer acetate reduces TH1, TH17 and B cell activity as well as sNfL, yet the addition of oral GlcNAc dose-dependently lowered serum IFNγ, IL-17, IL-6 and NfL. Oral GlcANc also dose-dependently reduced serum levels of the anti-inflammatory cytokine IL-10, which is increased in the brain of MS patients. 30% of treated patients displayed confirmed improvement in neurological disability, with an average EDSS score decrease of 0.52 points. CONCLUSIONS: Oral GlcNAc inhibits inflammation and neurodegeneration markers in MS patients despite concurrent immunomodulation by glatiramer acetate. Blinded studies are required to investigate GlcNAc's potential to control residual brain inflammation, myelin repair and neurodegeneration in MS.

Immunological Predictors of Dimethyl Fumarate-Induced Lymphopenia.

Treatment with dimethyl fumarate (DMF) leads to lymphopenia and infectious complications in a subset of patients with multiple sclerosis (MS). Here, we aimed to reveal immune markers of DMF-associated lymphopenia. This prospective observational study longitudinally assessed 31 individuals with MS by single-cell mass cytometry before and after 12 and 48 weeks of DMF therapy. Employing a neural network-based representation learning approach, we identified a CCR4-expressing T helper cell population negatively associated with relevant lymphopenia. CCR4-expressing T helper cells represent a candidate prognostic biomarker for the development of relevant lymphopenia in patients undergoing DMF treatment. ANN NEUROL 2022;91:676-681.

A New Advanced MRI Biomarker for Remyelinated Lesions in Multiple Sclerosis.

OBJECTIVES: Neuropathological studies have shown that multiple sclerosis (MS) lesions are heterogeneous in terms of myelin/axon damage and repair as well as iron content. However, it remains a challenge to identify specific chronic lesion types, especially remyelinated lesions, in vivo in patients with MS. METHODS: We performed 3 studies: (1) a cross-sectional study in a prospective cohort of 115 patients with MS and 76 healthy controls, who underwent 3 T magnetic resonance imaging (MRI) for quantitative susceptibility mapping (QSM), myelin water fraction (MWF), and neurite density index (NDI) maps. White matter (WM) lesions in QSM were classified into 5 QSM lesion types (iso-intense, hypo-intense, hyperintense, lesions with hypo-intense rims, and lesions with paramagnetic rim legions [PRLs]); (2) a longitudinal study of 40 patients with MS to study the evolution of lesions over 2 years; (3) a postmortem histopathology-QSM validation study in 3 brains of patients with MS to assess the accuracy of QSM classification to identify neuropathological lesion types in 63 WM lesions. RESULTS: At baseline, hypo- and isointense lesions showed higher mean MWF and NDI values compared to other QSM lesion types (p < 0.0001). Further, at 2-year follow-up, hypo-/iso-intense lesions showed an increase in MWF. Postmortem analyses revealed that QSM highly accurately identifies (1) fully remyelinated areas as hypo-/iso-intense (sensitivity = 88.89% and specificity = 100%), (2) chronic inactive lesions as hyperintense (sensitivity = 71.43% and specificity = 92.00%), and (3) chronic active/smoldering lesions as PRLs (sensitivity = 92.86% and specificity = 86.36%). INTERPRETATION: These results provide the first evidence that it is possible to distinguish chronic MS lesions in a clinical setting, hereby supporting with new biomarkers to develop and assess remyelinating treatments. ANN NEUROL 2022;92:486-502.

Intrathecal IgM Synthesis Is Associated with Spinal Cord Manifestation and Neuronal Injury in Early MS.

OBJECTIVE: Intrathecal Immunoglobulin M synthesis (IgMIntrathecal Fraction (IF) + ) and spinal MRI lesions are both strong independent predictors of higher disease activity and severity in multiple sclerosis (MS). We investigated whether IgMIF + is associated with spinal cord manifestation and higher neuroaxonal damage in early MS. METHODS: In 122 patients with a first demyelinating event associations between (1) spinal versus (vs) non-spinal clinical syndrome (2) spinal vs cerebral T2-weighted (T2w) and (3) contrast-enhancing (CE) lesion counts with IgGIF + (vs IgGIF - ) or IgMIF + (vs IgMIF - ) were investigated by logistic regression adjusted for age and sex, respectively. For serum neurofilament light chain (sNfL) analysis patients were categorized for presence or absence of oligoclonal IgG bands (OCGB), IgGIF and IgMIF (>0% vs 0%, respectively): (1) OCGB- /IgGIF - /IgMIF - ; (2) OCGB+ /IgGIF - /IgMIF - ; (3) OCGB+ /IgGIF + /IgMIF - ; and (4) OCGB+ /IgGIF + /IgMIF + . Associations between categories 2 to 4 vs category 1 with sNfL concentrations were analyzed by robust linear regression, adjusted for sex and MRI parameters. RESULTS: Patients with a spinal syndrome had a 8.36-fold higher odds of IgMIF + (95%CI 3.03-23.03; p < 0.01). Each spinal T2w lesion (odds Ratio 1.39; 1.02-1.90; p = 0.037) and CE lesion (OR 2.73; 1.22-6.09; p = 0.014) was associated with an increased risk of IgMIF + (but not of IgGIF + ); this was not the case for cerebral lesions. OCGB+ /IgGIF + /IgMIF + category patients showed highest sNfL levels (estimate:1.80; 0.55-3.06; p < 0.01). INTERPRETATION: Intrathecal IgM synthesis is strongly associated with spinal manifestation and independently more pronounced neuroaxonal injury in early MS, suggesting a distinct clinical phenotype and pathophysiology. ANN NEUROL 2022;91:814-820.

Stratifying the Presymptomatic Phase of Genetic Frontotemporal Dementia by Serum NfL and pNfH: A Longitudinal Multicentre Study.

OBJECTIVE: Although the presymptomatic stages of frontotemporal dementia (FTD) provide a unique chance to delay or even prevent neurodegeneration by early intervention, they remain poorly defined. Leveraging a large multicenter cohort of genetic FTD mutation carriers, we provide a biomarker-based stratification and biomarker cascade of the likely most treatment-relevant stage within the presymptomatic phase: the conversion stage. METHODS: We longitudinally assessed serum levels of neurofilament light (NfL) and phosphorylated neurofilament heavy (pNfH) in the Genetic FTD Initiative (GENFI) cohort (n = 444), using single-molecule array technique. Subjects comprised 91 symptomatic and 179 presymptomatic subjects with mutations in the FTD genes C9orf72, GRN, or MAPT, and 174 mutation-negative within-family controls. RESULTS: In a biomarker cascade, NfL increase preceded the hypothetical clinical onset by 15 years and concurred with brain atrophy onset, whereas pNfH increase started close to clinical onset. The conversion stage was marked by increased NfL, but still normal pNfH levels, while both were increased at the symptomatic stage. Intra-individual change rates were increased for NfL at the conversion stage and for pNfH at the symptomatic stage, highlighting their respective potential as stage-dependent dynamic biomarkers within the biomarker cascade. Increased NfL levels and NfL change rates allowed identification of presymptomatic subjects converting to symptomatic disease and capture of proximity-to-onset. We estimate stage-dependent sample sizes for trials aiming to decrease neurofilament levels or change rates. INTERPRETATION: Blood NfL and pNfH provide dynamic stage-dependent stratification and, potentially, treatment response biomarkers in presymptomatic FTD, allowing demarcation of the conversion stage. The proposed biomarker cascade might pave the way towards a biomarker-based precision medicine approach to genetic FTD. ANN NEUROL 2022;91:33-47.

Association of serum neurofilament light with microglial activation in multiple sclerosis.

BACKGROUND: Translocator protein (TSPO)-PET and neurofilament light (NfL) both report on brain pathology, but their potential association has not yet been studied in multiple sclerosis (MS) in vivo. We aimed to evaluate the association between serum NfL (sNfL) and TSPO-PET-measurable microglial activation in the brain of patients with MS. METHODS: Microglial activation was detected using PET and the TSPO-binding radioligand [11C]PK11195. Distribution volume ratio (DVR) was used to evaluate specific [11C]PK11195-binding. sNfL levels were measured using single molecule array (Simoa). The associations between [11C]PK11195 DVR and sNfL were evaluated using correlation analyses and false discovery rate (FDR) corrected linear regression modelling. RESULTS: 44 patients with MS (40 relapsing-remitting and 4 secondary progressive) and 24 age-matched and sex-matched healthy controls were included. In the patient group with elevated brain [11C]PK11195 DVR (n=19), increased sNfL associated with higher DVR in the lesion rim (estimate (95% CI) 0.49 (0.15 to 0.83), p(FDR)=0.04) and perilesional normal appearing white matter (0.48 (0.14 to 0.83), p(FDR)=0.04), and with a higher number and larger volume of TSPO-PET-detectable rim-active lesions defined by microglial activation at the plaque edge (0.46 (0.10 to 0.81), p(FDR)=0.04 and 0.50 (0.17 to 0.84), p(FDR)=0.04, respectively). Based on the multivariate stepwise linear regression model, the volume of rim-active lesions was the most relevant factor affecting sNfL. CONCLUSIONS: Our demonstration of an association between microglial activation as measured by increased TSPO-PET signal, and elevated sNfL emphasises the significance of smouldering inflammation for progression-promoting pathology in MS and highlights the role of rim-active lesions in promoting neuroaxonal damage.

Visual function resists early neurodegeneration in the visual system in primary progressive multiple sclerosis.

BACKGROUND: Neurodegeneration in multiple sclerosis (MS) affects the visual system but dynamics and pathomechanisms over several years especially in primary progressive MS (PPMS) are not fully understood. METHODS: We assessed longitudinal changes in visual function, retinal neurodegeneration using optical coherence tomography, MRI and serum NfL (sNfL) levels in a prospective PPMS cohort and matched healthy controls. We investigated the changes over time, correlations between outcomes and with loss of visual function. RESULTS: We followed 81 patients with PPMS (mean disease duration 5.9 years) over 2.7 years on average. Retinal nerve fibre layer thickness (RNFL) was reduced in comparison with controls (90.1 vs 97.8 µm; p<0.001). Visual function quantified by the area under the log contrast sensitivity function (AULCSF) remained stable over a continuous loss of RNFL (0.46 µm/year, 95% CI 0.10 to 0.82; p=0.015) up until a mean turning point of 91 µm from which the AULCSF deteriorated. Intereye RNFL asymmetry above 6 µm, suggestive of subclinical optic neuritis, occurred in 15 patients and was related to lower AULCSF but occurred also in 5 out of 44 controls. Patients with an AULCSF progression had a faster increase in Expanded Disability Status Scale (beta=0.17/year, p=0.043). sNfL levels were elevated in patients (12.2 pg/mL vs 8.0 pg/mL, p<0.001), but remained stable during follow-up (beta=-0.14 pg/mL/year, p=0.291) and were not associated with other outcomes. CONCLUSION: Whereas neurodegeneration in the anterior visual system is already present at onset, visual function is not impaired until a certain turning point. sNfL is not correlated with structural or functional impairment in the visual system.