To identify pathogenetic markers and potential drivers of different lesion types in the white matter (WM) of patients with progressive multiple sclerosis (PMS), we sequenced RNA from 73 different WM areas. Compared to 25 WM controls, 6713 out of 18,609 genes were significantly differentially expressed in MS tissues (FDR < 0.05). A computational systems medicine analysis was performed to describe the MS lesion endophenotypes. The cellular source of specific molecules was examined by RNAscope, immunohistochemistry, and immunofluorescence. To examine common lesion specific mechanisms, we performed de novo network enrichment based on shared differentially expressed genes (DEGs), and found TGFβ-R2 as a central hub. RNAscope revealed astrocytes as the cellular source of TGFβ-R2 in remyelinating lesions. Since lesion-specific unique DEGs were more common than shared signatures, we examined lesion-specific pathways and de novo networks enriched with unique DEGs. Such network analysis indicated classic inflammatory responses in active lesions; catabolic and heat shock protein responses in inactive lesions; neuronal/axonal specific processes in chronic active lesions. In remyelinating lesions, de novo analyses identified axonal transport responses and adaptive immune markers, which was also supported by the most heterogeneous immunoglobulin gene expression. The signature of the normal-appearing white matter (NAWM) was more similar to control WM than to lesions: only 465 DEGs differentiated NAWM from controls, and 16 were unique. The upregulated marker CD26/DPP4 was expressed by microglia in the NAWM but by mononuclear cells in active lesions, which may indicate a special subset of microglia before the lesion develops, but also emphasizes that omics related to MS lesions should be interpreted in the context of different lesions types. While chronic active lesions were the most distinct from control WM based on the highest number of unique DEGs (n = 2213), remyelinating lesions had the highest gene expression levels, and the most different molecular map from chronic active lesions. This may suggest that these two lesion types represent two ends of the spectrum of lesion evolution in PMS. The profound changes in chronic active lesions, the predominance of synaptic/neural/axonal signatures coupled with minor inflammation may indicate end-stage irreversible molecular events responsible for this less treatable phase.

ObjectivesIn a prospective phase IV trial of the first-line oral treatment dimethyl fumarate (DMF), we examined dynamics of neurofilament light (NFL) chain in serum, plasma and cerebrospinal fluid (CSF) samples collected over 12 months from relapsing-remitting multiple sclerosis (RRMS) patients. NFL changes were related to disease activity.MethodsWe examined NFL levels by single-molecule array in 88 CSF, 348 plasma and 131 sera from treatment-naïve RRMS patients (n=52), healthy controls (n=23) and a placebo group matched by age, sex and NFL (n=52). Plasma/sera were collected at baseline, and 1, 3, 6 and 12 months after DMF. CSF samples were collected at baseline and 12 months after DMF.ResultsNFL concentration in CSF, plasma and serum correlated highly (p<0.0001 for all), but plasma levels were only 76.9% of paired serum concentration. After 12 months of DMF treatment, NFL concentration decreased by 73%, 69% and 55% in the CSF, serum and plasma (p<0.0001, respectively). Significant reduction in blood was observed after 6 and 12 months treatment compared with baseline (p<0.01 and p<0.0001, respectively) and to placebo (p<0.0001). Patients with NFL above the 807.5 pg/mL cut-off in CSF had 5.0-times relative risk of disease activity (p<0.001).ConclusionsThis study provides Class II evidence that first-line DMF reduces NFL in both blood and CSF after 6 months and normalises CSF levels in 73% of patients. High NFL concentration in CSF after a year reflected disease activity. NFL levels were higher in serum than in plasma, which should be considered when NFL is used as a biomarker.

BackgroundThe cuprizone (CPZ) model of multiple sclerosis (MS) was used to identify microRNAs (miRNAs) related to in vivo de- and remyelination. We further investigated the role of miR-146a in miR-146a-deficient (KO) mice: this miRNA is differentially expressed in MS lesions and promotes differentiation of oligodendrocyte precursor cells (OPCs) during remyelination, but its role has not been examined during demyelination.MethodsMicroRNAs were examined by Agilent Mouse miRNA Microarray in the corpus callosum during CPZ-induced demyelination and remyelination. Demyelination, axonal loss, changes in number of oligodendrocytes, OPCs, and macrophages/microglia was compared by histology/immunohistochemistry between KO and WT mice. Differential expression of target genes and proteins of miR-146a was analyzed in the transcriptome (4 × 44K Agilent Whole Mouse Genome Microarray) and proteome (liquid chromatography tandem mass spectrometry) of CPZ-induced de- and remyelination in WT mice. Levels of proinflammatory molecules in the corpus callosum were compared in WT versus KO mice by Meso Scale Discovery multiplex protein analysis.ResultsmiR-146a was increasingly upregulated during CPZ-induced de- and remyelination. The absence of miR-146a in KO mice protected against demyelination, axonal loss, body weight loss, and atrophy of thymus and spleen. The number of CNP+ oligodendrocytes was increased during demyelination in the miR-146a KO mice, while there was a trend of increased number of NG2+ OPCs in the WT mice. miR-146a target genes, SNAP25 and SMAD4, were downregulated in the proteome of demyelinating corpus callosum in WT mice. Higher levels of SNAP25 were measured by ELISA in the corpus callosum of miR-146a KO mice, but there was no difference between KO and WT mice during demyelination. Multiplex protein analysis of the corpus callosum lysate revealed upregulated TNF-RI, TNF-RII, and CCL2 in the WT mice in contrast to KO mice. The number of Mac3+ and Iba1+ macrophages/microglia was reduced in the demyelinating corpus callosum of the KO mice.ConclusionDuring demyelination, absence of miR-146a reduced inflammatory responses, demyelination, axonal loss, the number of infiltrating macrophages, and increased the number of myelinating oligodendrocytes. The number of OPCs was slightly higher in the WT mice during remyelination, indicating a complex role of miR-146a during in vivo de- and remyelination.