The TAM family of receptor protein tyrosine kinases comprises three known members, namely Tyro3, Axl, and Mer. These receptors are widely expressed in the nervous system, including by oligodendrocytes, the cell type responsible for myelinating the CNS. We examined the potential role of the TAM family and of their principle cognate ligand, Gas6 (growth arrest gene 6), in modulating the phenotype of the cuprizone model of demyelination. We found that the expression profiles of Axl, Mer, and Gas6 mRNA were increased in the corpus callosum in a temporal profile correlating with the increased migration and proliferation of microglia/macrophages in this model. In contrast, expression of Tyro3 decreased, correlating with the loss of oligodendrocytes. Gas6 both promoted in vitro survival of oligodendrocytes (39.3 Ϯ 3.1 vs 11.8 Ϯ 2.4%) and modulated markers of activation in purified cultures of microglia (tumor necrosis factor ␣ mRNA expression was reduced ϳ48%). In Gas6 Ϫ/Ϫ mice subjected to cuprizone-challenge, demyelination was greater than in control mice, within the rostral region of the corpus callosum, as assessed by luxol fast blue staining (myelination reduced by 36%) and by ultrastructural analysis. An increased loss of Gst-(glutathione S-transferase-)-positive oligodendrocytes was also identified throughout the corpus callosum of Gas6 Ϫ/Ϫ mice. Microglial marker expression (ionized calcium-binding adapter molecule 1) was increased in Gas6 Ϫ/Ϫ mice but was restricted to the rostral corpus callosum. Therefore, TAM receptor activation and regulation can independently influence both oligodendrocyte survival and the microglial response after CNS damage.

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Objective:To investigate pregnancy-related disease activity in a contemporary multiple sclerosis (MS) cohort.Methods:Using data from the MSBase Registry, we included pregnancies conceived after 31 Dec 2010 from women with relapsing-remitting MS or clinically isolated syndrome. Predictors of intrapartum relapse, and postpartum relapse and disability progression were determined by clustered logistic regression or Cox regression analyses.Results:We included 1998 pregnancies from 1619 women with MS. Preconception annualized relapse rate (ARR) was 0.29 (95% CI 0.27-0.32), fell to 0.19 (0.14-0.24) in third trimester, and increased to 0.59 (0.51-0.67) in early postpartum. Among women who used fingolimod or natalizumab, ARR before pregnancy was 0.37 (0.28-0.49) and 0.29 (0.22-0.37), respectively, and increased during pregnancy. Intrapartum ARR decreased with preconception dimethyl fumarate use. ARR spiked after delivery across all DMT groups. Natalizumab continuation into pregnancy reduced the odds of relapse during pregnancy (OR 0.76 per month [0.60-0.95], p=0.017). DMT re-initiation with natalizumab protected against postpartum relapse (HR 0.11 [0.04-0.32], p<0.0001). Breastfeeding women were less likely to relapse (HR 0.61 [0.41-0.91], p=0.016). 5.6% of pregnancies were followed by confirmed disability progression, predicted by higher relapse activity in pregnancy and postpartum.Conclusion:Intrapartum and postpartum relapse probabilities increased among women with MS after natalizumab or fingolimod cessation. In women considered to be at high relapse risk, use of natalizumab before pregnancy and continued up to 34 weeks gestation, with early re-initiation after delivery is an effective option to minimize relapse risks. Strategies of DMT use have to be balanced against potential fetal/neonatal complications.

Multiple sclerosis (MS) is a chronic relapsing-remitting inflammatory disease of the central nervous system characterized by oligodendrocyte damage, demyelination and neuronal death. Genetic association studies have shown a 2-fold or greater prevalence of the HLA-DRB1*1501 allele in the MS population compared with normal Caucasians. In discovery cohorts of Australasian patients with MS (total 2941 patients and 3008 controls), we examined the associations of 12 functional polymorphisms of P2X7, a microglial/macrophage receptor with proinflammatory effects when activated by extracellular adenosine triphosphate (ATP). In discovery cohorts, rs28360457, coding for Arg307Gln was associated with MS and combined analysis showed a 2-fold lower minor allele frequency compared with controls (1.11% for MS and 2.15% for controls, P = 0.0000071). Replication analysis of four independent European MS case-control cohorts (total 2140 cases and 2634 controls) confirmed this association [odds ratio (OR) = 0.69, P = 0.026]. A meta-analysis of all Australasian and European cohorts indicated that Arg307Gln confers a 1.8-fold protective effect on MS risk (OR = 0.57, P = 0.0000024). Fresh human monocytes heterozygous for Arg307Gln have >85% loss of 'pore' function of the P2X7 receptor measured by ATP-induced ethidium uptake. Analysis shows Arg307Gln always occurred with 270His suggesting a single 307Gln-270His haplotype that confers dominant negative effects on P2X7 function and protection against MS. Modeling based on the homologous zP2X4 receptor showed Arg307 is located in a region rich in basic residues located only 12 Å from the ligand binding site. Our data show the protective effect against MS of a rare genetic variant of P2RX7 with heterozygotes showing near absent proinflammatory 'pore' function.

The limited efficacy of glucocorticoids (GCs) during therapy of acute relapses in multiple sclerosis (MS) leads to long-term disability. We investigated the potential of vitamin D (VD) to enhance GC efficacy and the mechanisms underlying this VD/GC interaction. In vitro, GC receptor (GR) expression levels were quantified by ELISA and induction of T cell apoptosis served as a functional readout to assess synergistic 1,25(OH) 2 D 3 (1,25D)/GC effects. Experimental autoimmune encephalomyelitis (MOG 35–55 EAE) was induced in mice with T cell-specific GR or mTORc1 deficiency. 25(OH)D (25D) levels were determined in two independent cohorts of MS patients with stable disease or relapses either responsive or resistant to GC treatment (initial cohort: n = 110; validation cohort: n = 85). Gene expression of human CD8 + T cells was analyzed by microarray ( n = 112) and correlated with 25D serum levels. In vitro, 1,25D upregulated GR protein levels, leading to increased GC-induced T cell apoptosis. 1,25D/GC combination therapy ameliorated clinical EAE course more efficiently than respective monotherapies, which was dependent on GR expression in T cells. In MS patients from two independent cohorts, 25D deficiency was associated with GC-resistant relapses. Mechanistic studies revealed that synergistic 1,25D/GC effects on apoptosis induction were mediated by the mTOR but not JNK pathway. In line, 1,25D inhibited mTORc1 activity in murine T cells, and low 25D levels in humans were associated with a reduced expression of mTORc1 inhibiting tuberous sclerosis complex 1 in CD8 + T cells. GR upregulation by 1,25D and 1,25D/GC synergism in vitro and therapeutic efficacy in vivo were abolished in animals with a T cell-specific mTORc1 deficiency. Specific inhibition of mTORc1 by everolimus increased the efficacy of GC in EAE. 1,25D augments GC-mediated effects in vitro and in vivo in a T cell-specific, GR-dependent manner via mTORc1 inhibition. These data may have implications for improvement of anti-inflammatory GC therapy. Electronic supplementary material The online version of this article (10.1007/s00401-019-02018-8) contains supplementary material, which is available to authorized users.

In multiple sclerosis, inflammatory axonal injury is a key pathological mechanism responsible for the development of progressive neurological dysfunction. The injured axon represents a therapeutic target in this disease; however, therapeutic trials of neuroprotective candidates will initially require preclinical testing in an animal model of inflammatory axonal injury and subsequently the development of a reliable paraclinical measure of axonal degeneration in humans. In the present study, we demonstrate the validity of serum phosphorylated neurofilament H (pNF-H) as a marker of axonal injury in murine experimental autoimmune encephalomyelitis (EAE). At the time of maximum disease severity (EAE day 22), the average serum pNF-H level reached 5.7 ng/ml, correlating significantly with the EAE paraplegia score (r = 0.75, P < 0.001). On average, 40% of axons in the spinal cord were lost in EAE, and serum pNF-H levels were highly correlated with axon loss (r = 0.8, P < 0.001). Axonal injury was a severe and acute event, insofar as serum pNF-H levels were not significantly elevated at early (EAE day 12) or late (EAE days 35 and 50) disease time points. Our results demonstrate that acute inflammatory axonal injury is a pathological feature of murine MOG(35-55) EAE, indicating that this model may mirror the acute pathological events in active multiple sclerosis lesions. Furthermore, we have validated the serum pNF-H assay as an unbiased measurement of axonal injury in EAE, facilitating rapid screening of potential neuroprotective therapies in this model.

Biomarkers of axonal degeneration have the potential to improve our capacity to predict and monitor neurological outcome in multiple sclerosis (MS) patients. Neurofilament proteins, one of the major proteins expressed within neurons and axons, have been detected in cerebrospinal fluid and blood samples from MS patients and are now being actively investigated for their utility as prognostic indicators of disease progression in MS. In this paper, we summarize the current literature on neurofilament structure, assembly, and degeneration and discuss their potential utility as biomarkers for monitoring neurological decline in MS. We also discuss the need to further develop sensitive methods for assaying neurofilaments in blood to improve clinical applicability.

BackgroundNeuroinflammation regulates both disease pathogenesis and repair in multiple sclerosis. In early multiple sclerosis lesion development, neuroinflammation causes demyelination and axonal injury, the likely final common determinant of disability. Here we report the identification of a novel neuroinflammatory mediator, Disabled-2 (Dab2). Dab2 is an intracellular adaptor protein with previously unknown function in the central nervous system.ResultsWe report that Dab2 is up-regulated in lesional macrophages/microglia in the spinal cord in murine experimental autoimmune encephalomyelitis, a model of multiple sclerosis. We demonstrate that dab2 expression is positively correlated with experimental autoimmune encephalomyelitis disease severity during the acute disease phase. Furthermore, dab2-deficient mice have a less severe experimental autoimmune encephalomyelitis disease course and suffer less neuroinflammation and less axonal injury than their wild-type littermates. We demonstrate that dab2 expression is strongly associated with the expression of inducible nitric oxide synthase. We further demonstrate that Dab2 is expressed at the protein level by macrophages in early acute human multiple sclerosis lesions and that this correlates with axonal injury.ConclusionsTogether, these results suggest that endogenous Dab2 exacerbates central nervous system inflammation, potentially acting to up-regulate reactive oxygen species expression in macrophages and microglia, and that it is of potential pathogenic relevance in Multiple Sclerosis.