Shared imaging markers of fatigue across multiple sclerosis, aquaporin-4 antibody neuromyelitis optica spectrum disorder and MOG antibody disease.

Fatigue is frequently reported by patients with multiple sclerosis, aquaporin-4-antibody neuromyelitis optica spectrum disorder and myelin-oligodendrocyte-glycoprotein antibody disease; thus they could share a similar pathophysiological mechanism. In this cross-sectional cohort study, we assessed the association of fatigue with resting-state functional MRI, diffusion and structural imaging measures across these three disorders. Sixteen patients with multiple sclerosis, 17 with aquaporin-4-antibody neuromyelitis optica spectrum disorder and 17 with myelin-oligodendrocyte-glycoprotein antibody disease assessed, outside of relapses, at the Oxford Neuromyelitis Optica Service underwent Modified Fatigue Impact Scale, Hospital Anxiety and Depression Scale and Expanded Disability Status Scale scoring. A 3T brain and spinal cord MRI was used to derive cortical, deep grey and white matter volumetrics, lesions volume, fractional anisotropy, brain functional connectivity metrics, cervical spinal cord cross-sectional area, spinal cord magnetic transfer ratio and average functional connectivity between the ventral and the dorsal horns of the cervical cord. Linear relationships between MRI measures and total-, cognitive- and physical-fatigue scores were assessed. All analyses were adjusted for correlated clinical regressors. No significant differences in baseline clinical characteristics, fatigue, depression and anxiety questionnaires and disability measures were seen across the three diseases, except for older age in patients with aquaporin-4-antibody neuromyelitis optica spectrum disorder (P = 0.0005). In the total cohort, median total-fatigue score was 35.5 (range 3-72), and 42% of patients were clinically fatigued. A positive correlation existed between the total-fatigue score and functional connectivity of the executive/fronto-temporal network in the in left middle temporal gyrus (P = 0.033) and between the physical-fatigue score and functional connectivity of the sensory-motor network (P = 0.032) in both pre- and post-central gyri. A negative relationship was found between the total-fatigue score and functional connectivity of the salience network (P = 0.023) and of the left fronto-parietal network (P = 0.026) in the right supramarginal gyrus and left superior parietal lobe. No clear relationship between fatigue subscores and the average functional connectivity of the spinal cord was found. Cognitive-fatigue scores were positively associated with white matter lesion volume (P = 0.018) and negatively associated with white matter fractional anisotropy (P = 0.032). Structural, diffusion and functional connectivity alterations were not influenced by the disease group. Functional and structural imaging metrics associated with fatigue relate to brain rather than spinal cord abnormalities. Salience and sensory-motor networks alterations in relation to fatigue might indicate a disconnection between the perception of the interior body state and activity and the actual behavioural responses and performances (reversible or irreversible). Future research should focus on functional rehabilitative strategies.

Acute Inflammatory Diseases of the Central Nervous System After SARS-CoV-2 Vaccination.

BACKGROUND AND OBJECTIVES: Acute inflammatory CNS diseases include neuromyelitis optica spectrum disorders (NMOSDs) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD). Both MOGAD and acute disseminated encephalomyelitis (ADEM) have been reported after vaccination. Consequently, the mass SARS-CoV-2 vaccination program could result in increased rates of these conditions. We described the features of patients presenting with new acute CNS demyelination resembling NMOSDs or MOGAD within 8 weeks of SARS-CoV-2 vaccination. METHODS: The study included a prospective case series of patients referred to highly specialized NMOSD services in the UK from the introduction of SARS-CoV-2 vaccination program up to May 2022. Twenty-five patients presented with new optic neuritis (ON) and/or transverse myelitis (TM) ± other CNS inflammation within 8 weeks of vaccination with either AstraZeneca (ChAdOx1S) or Pfizer (BNT162b2) vaccines. Their clinical records and paraclinical investigations including MRI scans were reviewed. Serologic testing for antibodies to myelin oligodendrocyte glycoprotein (MOG) and aquaporin 4 (AQP4) was performed using live cell-based assays. Patients' outcomes were graded good, moderate, or poor based on the last clinical assessment. RESULTS: Of 25 patients identified (median age 38 years, 14 female), 12 (48%) had MOG antibodies (MOGIgG+), 2 (8%) had aquaporin 4 antibodies (AQP4IgG+), and 11 (44%) had neither. Twelve of 14 (86%) antibody-positive patients received the ChAdOx1S vaccine. MOGIgG+ patients presented most commonly with TM (10/12, 83%), frequently in combination with ADEM-like brain/brainstem lesions (6/12, 50%). Transverse myelitis was longitudinally extensive in 7 of the 10 patients. A peak in new MOGAD cases in Spring 2021 was attributable to postvaccine cases. Both AQP4IgG+ patients presented with brain lesions and TM. Four of 6 (67%) seronegative ChAdOx1S recipients experienced longitudinally extensive TM (LETM) compared with 1 of 5 (20%) of the BNT162b2 group, and facial nerve inflammation was reported only in ChAdOx1S recipients (2/5, 40%). Guillain-Barre syndrome was confirmed in 1 seronegative ChAdOx1S recipient and suspected in another. DISCUSSION: ChAdOx1S was associated with 12/14 antibody-positive cases, the majority MOGAD. MOGAD patients presented atypically, only 2 with isolated ON (1 after BNT162b2 vaccine) but with frequent ADEM-like brain lesions and LETM. Within the seronegative group, phenotypic differences were observed between ChAdOx1S and BNT162b2 recipients. These observations might support a causative role of the ChAdOx1S vaccine in inflammatory CNS disease and particularly MOGAD. Further study of this cohort could provide insights into vaccine-associated immunopathology.

Unmet Needs and Treatment of Relapsing-Remitting Multiple Sclerosis in Saudi Arabia: Focus on the Role of Ofatumumab.

Treatment-pattern data suggest that some patients with multiple sclerosis (MS) in the Kingdom of Saudi Arabia (KSA) may not be receiving optimal treatment. A virtual meeting of ten expert Saudi neurologists, held on October 23, 2020, discussed unmet needs in relapsing-remitting MS (RRMS), and the role of ofatumumab as a suitable treatment in the KSA. Multiple unmet needs were identified: poor quality of life, with high rates of depression and anxiety; a negative impact of MS on work ability; treatment choices that may compromise efficacy for safety or vice versa; inconvenient or complex dosage regimens; and limited access to patient education and support. Early use of highly effective disease-modifying treatments (DMTs) results in better patient outcomes than starting with less effective treatments and downstream escalation, but this strategy may be underutilized in the KSA. B cells are important in MS pathogenesis, and treatments targeting these may improve clinical outcomes. Ofatumumab differs from other B cell-depleting therapies, being a fully human monoclonal antibody that binds to CD20 at a completely separate site from the epitope bound by ocrelizumab, and being administered by subcutaneous injection. When compared with teriflunomide in two randomized, phase 3 clinical trials in patients with RRMS, ofatumumab was associated with significant reductions in annualized relapse rates, rates of confirmed disability worsening, and active lesions on magnetic resonance imaging. The incidence of adverse events, including serious infections, was similar with the two treatments. Ofatumumab is a valuable first- or second-line treatment option for RRMS in the KSA, particularly for patients who would benefit from highly effective DMTs early in the disease course, and for those who prefer the convenience of self-injection. Future research will clarify the position of ofatumumab in RRMS treatment, and comparative cost data may support the broad inclusion of ofatumumab in formularies across the KSA.

Evaluating the feasibility of a real world pharmacovigilance study (OPTIMISE:MS).

BACKGROUND: Clinical trial populations do not fully reflect routine practice. The power of routinely collected data to inform clinical practice is increasingly recognised. METHODS: The OPTIMISE:MS pharmacovigilance study is a prospective, pragmatic observational study, conducted across 13 UK MS centres. Data were collected at the time of routine clinical visits. The first participant was recruited on 24th May 2019; data were extracted on 11th November 2021. RESULTS: 2112 participants were included (median age 44.0 years; 1570 (72%) female; 1981 (94%) relapsing-remitting MS). 639 (30%) were untreated at study entry, 205 (10%) taking interferon beta/copaxone, 1004 (47%) second/third generation DMT first line and 264 (13%) had escalated from a platform DMT. 342 clinical events were reported, of which 108 infections. There was an increased risk of adverse events in people taking second/third generation DMT (RR 3.45, 95%CI 1.57-7.60, p<0.01 vs no DMT). Unadjusted Poisson regression demonstrated increased incident adverse events in people taking natalizumab (IRR 5.28, 95%CI 1.41-19.74, p<0.05), ocrelizumab (IRR 3.24, 95%CI 1.22-8.62, p<0.05), and GA biosimilar (Brabio) (IRR 4.89, 95%CI 1.31-18.21, p<0.05) vs no DMT. CONCLUSIONS: Routinely collected healthcare data can be used to evaluate DMT safety in people with MS. These data highlight the potential of pragmatic studies to guide understanding of risks and benefits associated with DMT.

OPTIMISE: MS study protocol: a pragmatic, prospective observational study to address the need for, and challenges with, real world pharmacovigilance in multiple sclerosis.

INTRODUCTION: The power of 'real world' data to improve our understanding of the clinical aspects of multiple sclerosis (MS) is starting to be realised. Disease modifying therapy (DMT) use across the UK is driven by national prescribing guidelines. As such, the UK provides an ideal country in which to gather MS outcomes data. A rigorously conducted observational study with a focus on pharmacovigilance has the potential to provide important data to inform clinicians and patients while testing the reliability of estimates from pivotal trials when applied to patients in the UK. METHODS AND ANALYSIS: The primary aim of this study is to characterise the incidence and compare the risk of serious adverse events in people with MS treated with DMTs. The OPTIMISE:MS database enables electronic data capture and secure data transfer. Selected clinical data, clinical histories and patient-reported outcomes are collected in a harmonised fashion across sites at the time of routine clinical visits. The first patient was recruited to the study on 24 May 2019. As of January 2021, 1615 individuals have baseline data recorded; follow-up data are being captured and will be reported in due course. ETHICS AND DISSEMINATION: This study has ethical permission (London City and East; Ref 19/LO/0064). Potential concerns around data storage and sharing are mitigated by the separation of identifiable data from all other clinical data, and limiting access to any identifiable data. The results of this study will be disseminated via publication. Participants provide consent for anonymised data to be shared for further research use, further enhancing the value of the study.

An In-vivo 1H-MRS short-echo time technique at 7T: Quantification of metabolites in chronic multiple sclerosis and neuromyelitis optica brain lesions and normal appearing brain tissue.

Magnetic Resonance Spectroscopy (MRS) allows for the non-invasive quantification of neurochemicals and has the potential to differentiate between the pathologically distinct diseases, multiple sclerosis (MS) and AQP4Ab-positive neuromyelitis optica spectrum disorder (AQP4Ab-NMOSD). In this study we characterised the metabolite profiles of brain lesions in 11 MS and 4 AQP4Ab-NMOSD patients using an optimised MRS methodology at ultra-high field strength (7T) incorporating correction for T2 water relaxation differences between lesioned and normal tissue. MS metabolite results were in keeping with the existing literature: total N-acetylaspartate (NAA) was lower in lesions compared to normal appearing brain white matter (NAWM) with reciprocal findings for myo-Inositol. An unexpected subtlety revealed by our technique was that total NAA differences were likely driven by NAA-glutamate (NAAG), a ubiquitous CNS molecule with functions quite distinct from NAA though commonly quantified together with NAA in MRS studies as total NAA. Surprisingly, AQP4Ab-NMOSD showed no significant differences for total NAA, NAA, NAAG or myo-Inositol between lesion and NAWM sites, nor were there any differences between MS and AQP4Ab-NMOSD for a priori hypotheses. Post-hoc testing revealed a significant correlation between NAWM Ins:NAA and disability (as measured by EDSS) for disease groups combined, driven by the AP4Ab-NMOSD group. Utilising an optimised MRS methodology, our study highlights some under-explored subtleties in MRS profiles, such as the absence of myo-Inositol concentration differences in AQP4Ab-NMOSD brain lesions versus NAWM and the potential influence of NAAG differences between lesions and normal appearing white matter in MS.

Foveal changes in aquaporin-4 antibody seropositive neuromyelitis optica spectrum disorder are independent of optic neuritis and not overtly progressive.

BACKGROUND AND PURPOSE: Foveal changes were reported in aquaporin-4 antibody (AQP4-Ab) seropositive neuromyelitis optica spectrum disorder (NMOSD) patients; however, it is unclear whether they are independent of optic neuritis (ON), stem from subclinical ON or crossover from ON in fellow eyes. Fovea morphometry and a statistical classification approach were used to investigate if foveal changes in NMOSD are independent of ON and progressive. METHODS: This was a retrospective longitudinal study of 27 AQP4-IgG + NMOSD patients (49 eyes; 15 ON eyes and 34 eyes without a history of ON [NON eyes]), follow-up median (first and third quartile) 2.32 (1.33-3.28), and 38 healthy controls (HCs) (76 eyes), follow-up median (first and third quartile) 1.95 (1.83-2.54). The peripapillary retinal nerve fibre layer thickness and the volume of combined ganglion cell and inner plexiform layer as measures of neuroaxonal damage from ON were determined by optical coherence tomography. Nineteen foveal morphometry parameters were extracted from macular optical coherence tomography volume scans. Data were analysed using orthogonal partial least squares discriminant analysis and linear mixed effects models. RESULTS: At baseline, foveal shape was significantly altered in ON eyes and NON eyes compared to HCs. Discriminatory analysis showed 81% accuracy distinguishing ON vs. HCs and 68% accuracy in NON vs. HCs. NON eyes were distinguished from HCs by foveal shape parameters indicating widening. Orthogonal partial least squares discriminant analysis discriminated ON vs. NON with 76% accuracy. In a follow-up of 2.4 (20.85) years, no significant time-dependent foveal changes were found. CONCLUSION: The parafoveal area is altered in AQP4-Ab seropositive NMOSD patients suggesting independent neuroaxonal damage from subclinical ON. Longer follow-ups are needed to confirm the stability of the parafoveal structure over time.

Systematic approach to selecting licensed drugs for repurposing in the treatment of progressive multiple sclerosis.

OBJECTIVE: To establish a rigorous, expert-led, evidence-based approach to the evaluation of licensed drugs for repurposing and testing in clinical trials of people with progressive multiple sclerosis (MS). METHODS: We long-listed licensed drugs with evidence of human safety, blood-brain barrier penetrance and demonstrable efficacy in at least one animal model, or mechanistic target, agreed by a panel of experts and people with MS to be relevant to the pathogenesis of progression. We systematically reviewed the preclinical and clinical literature for each compound, condensed this into a database of summary documents and short-listed drugs by scoring each one of them. Drugs were evaluated for immediate use in a clinical trial, and our selection was scrutinised by a final independent expert review. RESULTS: From a short list of 55 treatments, we recommended four treatments for immediate testing in progressive MS: R-α-lipoic acid, metformin, the combination treatment of R-α-lipoic acid and metformin, and niacin. We also prioritised clemastine, lamotrigine, oxcarbazepine, nimodipine and flunarizine. CONCLUSIONS: We report a standardised approach for the identification of candidate drugs for repurposing in the treatment of progressive MS.

Amiloride does not protect retinal nerve fibre layer thickness in optic neuritis in a phase 2 randomised controlled trial.

BACKGROUND: Recent basic and clinical evidence suggests amiloride may be neuroprotective in multiple sclerosis (MS) through the blockade of the acid sensing ion channel (ASIC). OBJECTIVE: To examine the neuroprotective efficacy of amiloride in acute optic neuritis (ON). METHODS: A total of 48 patients were recruited to a phase 2, double blind, single site, randomised controlled trial. Scanning laser polarimetry (GDx) at 6 months was the primary outcome measure and optical coherence tomography (OCT) and visual and electrophysiological measures were secondary outcome measures. Participants aged 18-55 years, ≤28 days of onset of first episode unilateral ON, were randomised to amiloride (10 mg daily for 5 months) or placebo ( clinicaltrials.gov , NCT 01802489). RESULTS: Intention-to-treat (ITT) cohort consisted of 43 patients; 23 placebo and 20 amiloride. No significant drug-related adverse events occurred. No significant differences were found in GDx ( p = 0.840). Visual evoked potentials (VEP) were significantly prolonged in the amiloride group compared to placebo ( p = 0.004). All other secondary outcome measures showed no significant difference. Baseline analysis of OCT data demonstrated a significant pre-randomisation thinning of ganglion cell layer. CONCLUSION: Amiloride has not demonstrated any neuroprotective benefit within this trial paradigm, but future neuroprotective trials in ON should target the window of opportunity to maximise potential neuroprotective benefit.

Contribution of Fibrinogen to Inflammation and Neuronal Density in Human Traumatic Brain Injury.

Traumatic brain injury (TBI) is a leading cause of death and disability, particularly among the young. Despite this, no disease-specific treatments exist. Recently, blood-brain barrier disruption and parenchymal fibrinogen deposition have been reported in acute traumatic brain injury and in long-term survival; however, their contribution to the neuropathology of TBI remains unknown. The presence of fibrinogen-a well-documented activator of microglia/macrophages-may be associated with neuroinflammation, and neuronal/axonal injury. To test this hypothesis, cases of human TBI with survival times ranging from 12 h to 13 years (survival <2 months, n = 15; survival >1 year, n = 6) were compared with uninjured controls (n = 15). Tissue was selected from the frontal lobe, temporal lobe, corpus callosum, cingulate gyrus, and brainstem, and the extent of plasma protein (fibrinogen and immunoglobulin G [IgG]) deposition, microglial/macrophage activation (CD68 and ionized calcium-binding adapter molecule 1 [Iba-1] immunoreactivity), neuronal density, and axonal transport impairment (ß-amyloid precursor protein [ßAPP] immunoreactivity) were assessed. Quantitative analysis revealed a significant increase in parenchymal fibrinogen and IgG deposition following acute TBI compared with long-term survival and control. Fibrinogen, but not IgG, was associated with microglial/macrophage activation and a significant reduction in neuronal density. Perivascular fibrinogen deposition also was associated with microglial/macrophage clustering and accrual of ßAPP in axonal spheroids, albeit rarely. These findings mandate the future exploration of causal relationships between fibrinogen deposition, microglia/macrophage activation, and potential neuronal loss in acute TBI.

Brain lesion distribution criteria distinguish MS from AQP4-antibody NMOSD and MOG-antibody disease.

IMPORTANCE: Neuromyelitis optica spectrum disorders (NMOSD) can present with very similar clinical features to multiple sclerosis (MS), but the international diagnostic imaging criteria for MS are not necessarily helpful in distinguishing these two diseases. OBJECTIVE: This multicentre study tested previously reported criteria of '(1) at least 1 lesion adjacent to the body of the lateral ventricle and in the inferior temporal lobe; or (2) the presence of a subcortical U-fibre lesion or (3) a Dawson's finger-type lesion' in an independent cohort of relapsing-remitting multiple sclerosis (RRMS) and AQP4-ab NMOSD patients and also assessed their value in myelin oligodendrocyte glycoprotein (MOG)-ab positive and ab-negative NMOSD. DESIGN: Brain MRI scans were anonymised and scored on the criteria by 2 of 3 independent raters. In case of disagreement, the final opinion was made by the third rater. PARTICIPANTS: 112 patients with NMOSD (31 AQP4-ab-positive, 21 MOG-ab-positive, 16 ab-negative) or MS (44) were selected from 3 centres (Oxford, Strasbourg and Liverpool) for the presence of brain lesions. RESULTS: MRI brain lesion distribution criteria were able to distinguish RRMS with a sensitivity of 90.9% and with a specificity of 87.1% against AQP4-ab NMOSD, 95.2% against MOG-ab NMOSD and 87.5% in the heterogenous ab-negative NMOSD cohort. Over the whole NMOSD group, the specificity was 89.7%. CONCLUSIONS: This study suggests that the brain MRI criteria for differentiating RRMS from NMOSD are sensitive and specific for all phenotypes.

Amiloride Clinical Trial In Optic Neuritis (ACTION) protocol: a randomised, double blind, placebo controlled trial.

INTRODUCTION: Neurodegeneration is a widely accepted contributor to the development of long-term disability in multiple sclerosis (MS). While current therapies in MS predominantly target inflammation and reduce relapse rate they have been less effective at preventing long-term disability. The identification and evaluation of effective neuroprotective therapies within a trial paradigm are key unmet needs. Emerging evidence supports amiloride, a licenced diuretic, as a neuroprotective agent in MS through acid sensing ion channel blockade. Optic neuritis (ON) is a common manifestation of MS with correlates of inflammation and neurodegeneration measurable within the visual pathways. Amiloride Clinical Trial In Optic Neuritis (ACTION) will utilise a multimodal approach to assess the neuroprotective efficacy of amiloride in acute ON. METHODS AND ANALYSIS: 46 patients will be recruited within 28 days from onset of ON visual symptoms and randomised on a 1:1 basis to placebo or amiloride 10 mg daily. Double-blinded treatment groups will be balanced for age, sex and visual loss severity by a random-deterministic minimisation algorithm. The primary objective is to demonstrate that amiloride is neuroprotective in ON as assessed by scanning laser polarimetry of the peripapillary retinal nerve fibre layer (RNFL) thickness at 6 months in the affected eye compared to the unaffected eye at baseline. RNFL in combination with further retinal measures will also be assessed by optical coherence tomography. Secondary outcome measures on brain MRI will include cortical volume, diffusion-weighted imaging, resting state functional MRI, MR spectroscopy and magnetisation transfer ratio. In addition, high and low contrast visual acuity, visual fields, colour vision and electrophysiology will be assessed alongside quality of life measures. ETHICS AND DISSEMINATION: Ethical approval was given by the south central Oxford B research ethics committee (REC reference: 13/SC/0022). The findings from ACTION will be disseminated through peer-reviewed publications and at scientific conferences. TRIAL REGISTRATION NUMBER: EudraCT2012-004980-39, ClinicalTrials.gov Identifier: NCT01802489.

Overlapping CNS inflammatory diseases: differentiating features of NMO and MS.

Neuromyelitis optica (NMO) has long been considered as a variant of multiple sclerosis (MS) rather than a distinct disease. This concept changed with the discovery of serum antibodies (Ab) against aquaporin-4 (AQP4), which unequivocally differentiate NMO from MS. Patients who test positive for AQP4-Abs and present with optic neuritis (ON) and transverse myelitis (TM) are diagnosed with NMO and those who show an incomplete phenotype with isolated ON or longitudinally extensive TM (LETM) or less commonly brain/brainstem disease are referred to as NMO spectrum disorders (NMOSD). However, many patients, who have overlapping features of both NMO and MS, test negative for AQP4-Abs and may be difficult to definitively diagnose. This raises important practical issues, since NMO and MS respond differently to immunomodulatory treatment and have different prognoses. Here we review distinct features of AQP4-positive NMO and MS, which might then be useful in the diagnosis of antibody-negative overlap syndromes. We identify discriminators, which are related to demographic data (non-white origin, very late onset), clinical features (limited recovery from ON, bilateral ON, intractable nausea, progressive course of disability), laboratory results (cerebrospinal fluid (CSF) pleocytosis with eosinophils and/or neutrophils, oligoclonal bands, glial fibrillary acidic protein in the CSF) and imaging (LETM, LETM with T1 hypointensity, periependymal brainstem lesions, perivenous white matter lesions, Dawson's fingers, curved or S-shaped U-fibre juxtacortical lesions). We review the value of these discriminators and discuss the compelling need for new diagnostic markers in these two autoimmune demyelinating diseases of the central nervous system.

IL-21 and IL-21 receptor expression in lymphocytes and neurons in multiple sclerosis brain.

IL-17-producing CD4(+) T cells (Th-17) contribute to the pathogenesis of experimental autoimmune encephalomyelitis and are associated with active disease in multiple sclerosis (MS). In addition to IL-17, Th-17 cells can also express IL-21, IL-22, and IL-6 under Th-17-polarizing conditions (IL-6 and transforming growth factor-ß). In this study we investigated IL-21 and IL-21 receptor (IL-21R) expression in MS lesions by in situ hybridization and immunohistochemistry. We detected strongly IL-21(+) infiltrating cells predominantly in acute but also in chronic active white matter MS lesions in which IL-21 expression was restricted to CD4(+) cells. In contrast, IL-21R was much more broadly distributed on CD4(+), CD19(+), and CD8(+) lymphocytes but not major histocompatibility complex class-II(+) macrophages/microglia. Interestingly, in cortical areas we detected both IL-21 and IL-21R expression by neurons. These findings suggest role(s) for IL-21 in both the acute and chronic stages of MS via direct effects on T and B lymphocytes and, demonstrated for the first time, also on neurons.

Acid-sensing ion channel 1 is involved in both axonal injury and demyelination in multiple sclerosis and its animal model.

Although there is growing evidence for a role of excess intracellular cations, particularly calcium ions, in neuronal and glial cell injury in multiple sclerosis, as well as in non-inflammatory neurological conditions, the molecular mechanisms involved are not fully determined. We previously showed that the acid-sensing ion channel 1 which, when activated under the acidotic tissue conditions found in inflammatory lesions opens to allow influx of sodium and calcium ions, contributes to axonal injury in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. However, the extent and cellular distribution of acid-sensing ion channel 1 expression in neurons and glia in inflammatory lesions is unknown and, crucially, acid-sensing ion channel 1 expression has not been determined in multiple sclerosis lesions. Here we studied acute and chronic experimental autoimmune encephalomyelitis and multiple sclerosis spinal cord and optic nerve tissues to describe in detail the distribution of acid-sensing ion channel 1 and its relationship with neuronal and glial damage. We also tested the effects of amiloride treatment on tissue damage in the mouse models. We found that acid-sensing ion channel 1 was upregulated in axons and oligodendrocytes within lesions from mice with acute experimental autoimmune encephalomyelitis and from patients with active multiple sclerosis. The expression of acid-sensing ion channel 1 was associated with axonal damage as indicated by co-localization with the axonal injury marker beta amyloid precursor protein. Moreover, blocking acid-sensing ion channel 1 with amiloride protected both myelin and neurons from damage in the acute model, and when given either at disease onset or, more clinically relevant, at first relapse, ameliorated disability in mice with chronic-relapsing experimental autoimmune encephalomyelitis. Together these findings suggest that blockade of acid-sensing ion channel 1 has the potential to provide both neuro- and myelo-protective benefits in multiple sclerosis.

Opposing effects of HLA class I molecules in tuning autoreactive CD8+ T cells in multiple sclerosis.

The major known genetic risk factors in multiple sclerosis reside in the major histocompatibility complex (MHC) region. Although there is strong evidence implicating MHC class II alleles and CD4(+) T cells in multiple sclerosis pathogenesis, possible contributions from MHC class I genes and CD8(+) T cells are controversial. We have generated humanized mice expressing the multiple sclerosis-associated MHC class I alleles HLA-A(*)0301 (encoding human leukocyte antigen-A3 (HLA-A3)) and HLA-A(*)0201 (encoding HLA-A2) and a myelin-specific autoreactive T cell receptor (TCR) derived from a CD8(+) T cell clone from an individual with multiple sclerosis to study mechanisms of disease susceptibility. We demonstrate roles for HLA-A3-restricted CD8(+) T cells in induction of multiple sclerosis-like disease and for CD4(+) T cells in its progression, and we also define a possible mechanism for HLA-A(*)0201-mediated protection. To our knowledge, these data provide the first direct evidence incriminating MHC class I genes and CD8(+) T cells in the pathogenesis of human multiple sclerosis and reveal a network of MHC interactions that shape the risk of multiple sclerosis.

Interleukin-17 production in central nervous system-infiltrating T cells and glial cells is associated with active disease in multiple sclerosis.

Recent findings in the animal model for multiple sclerosis (MS), experimental autoimmune encephalomyelitis, implicate a novel CD4+ T-cell subset (TH17), characterized by the secretion of interleukin-17 (IL-17), in disease pathogenesis. To elucidate its role in MS, brain tissues from patients with MS were compared to controls. We detected expression of IL-17 mRNA (by in situ hybridization) and protein (by immunohistochemistry) in perivascular lymphocytes as well as in astrocytes and oligodendrocytes located in the active areas of MS lesions. Further, we found a significant increase in the number of IL-17+ T cells in active rather than inactive areas of MS lesions. Specifically, double immunofluorescence showed that IL-17 immunoreactivity was detected in 79% of T cells in acute lesions, 73% in active areas of chronic active lesions, but in only 17% of those in inactive lesions and 7% in lymph node control tissue. CD8+, as well as CD4+, T cells were equally immunostained for IL-17 in MS tissues. Interestingly, and in contrast to lymph node T cells, no perivascular T cells showed FoxP3 expression, a marker of regulatory T cells, at any stage of MS lesions. These observations suggest an enrichment of both IL-17+CD4+ and CD8+ T cells in active MS lesions as well as an important role for IL-17 in MS pathogenesis, with some remarkable differences from the experimental autoimmune encephalomyelitis model.

Acid-sensing ion channel-1 contributes to axonal degeneration in autoimmune inflammation of the central nervous system.

Multiple sclerosis is a neuroinflammatory disease associated with axonal degeneration. The neuronally expressed, proton-gated acid-sensing ion channel-1 (ASIC1) is permeable to Na+ and Ca2+, and excessive accumulation of these ions is associated with axonal degeneration. We tested the hypothesis that ASIC1 contributes to axonal degeneration in inflammatory lesions of the central nervous system (CNS). After induction of experimental autoimmune encephalomyelitis (EAE), Asic1-/- mice showed both a markedly reduced clinical deficit and reduced axonal degeneration compared to wild-type mice. Consistently with acidosis-mediated injury, pH measurements in the spinal cord of EAE mice showed tissue acidosis sufficient to open ASIC1. The acidosis-related protective effect of Asic1 disruption was also observed in nerve explants in vitro. Amiloride, a licensed and clinically safe blocker of ASICs, was equally neuroprotective in nerve explants and in EAE. Although ASICs are also expressed by immune cells, this expression is unlikely to explain the neuroprotective effect of Asic1 inactivation, as CNS inflammation was similar in wild-type and Asic1-/- mice. In addition, adoptive transfer of T cells from wild-type mice did not affect the protection mediated by Asic1 disruption. These results suggest that ASIC1 blockers could provide neuroprotection in multiple sclerosis.

Contactin regulates the current density and axonal expression of tetrodotoxin-resistant but not tetrodotoxin-sensitive sodium channels in DRG neurons.

Contactin, a glycosyl-phosphatidylinositol (GPI)-anchored predominantly neuronal cell surface glycoprotein, associates with sodium channels Nav1.2, Nav1.3 and Nav1.9, and enhances the density of these channels on the plasma membrane in mammalian expression systems. However, a detailed functional analysis of these interactions and of untested putative interactions with other sodium channel isoforms in mammalian neuronal cells has not been carried out. We examined the expression and function of sodium channels in small-diameter dorsal root ganglion (DRG) neurons from contactin-deficient (CNTN-/-) mice, compared to CNTN+/+ litter mates. Nav1.9 is preferentially expressed in isolectin B4 (IB4)-positive neurons and thus we used this marker to subdivide small-diameter DRG neurons. Using whole-cell patch-clamp recording, we observed a greater than two-fold reduction of tetrodotoxin-resistant (TTX-R) Nav1.8 and Nav1.9 current densities in IB4+ DRG neurons cultured from CNTN-/- vs. CNTN+/+ mice. Current densities for TTX-sensitive (TTX-S) sodium channels were unaffected. Contactin's effect was selective for IB4+ neurons as current densities for both TTX-R and TTX-S channels were not significantly different in IB4- DRG neurons from the two genotypes. Consistent with these results, we have demonstrated a reduction in Nav1.8 and Nav1.9 immunostaining on peripherin-positive unmyelinated axons in sciatic nerves from CNTN-/- mice but detected no changes in the expression for the two major TTX-S channels Nav1.6 and Nav1.7. These data provide evidence of a role for contactin in selectively regulating the cell surface expression and current densities of TTX-R but not TTX-S Na+ channel isoforms in nociceptive DRG neurons; this regulation could modulate the membrane properties and excitability of these neurons.

Sodium channels contribute to microglia/macrophage activation and function in EAE and MS.

Loss of axons is a major contributor to nonremitting deficits in the inflammatory demyelinating disease multiple sclerosis (MS). Based on biophysical studies showing that activity of axonal sodium channels can trigger axonal degeneration, recent studies have tested sodium channel-blocking drugs in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and have demonstrated a protective effect on axons. However, it is possible that, in addition to a direct effect on axons, sodium channel blockers may also interfere with inflammatory mechanisms. We therefore examined the novel hypothesis that sodium channels contribute to activation of microglia and macrophages in EAE and acute MS lesions. In this study, we demonstrate a robust increase of sodium channel Nav1.6 expression in activated microglia and macrophages in EAE and MS. We further demonstrate that treatment with the sodium channel blocker phenytoin ameliorates the inflammatory cell infiltrate in EAE by 75%. Supporting a role for sodium channels in microglial activation, we show that tetrodotoxin, a specific sodium channel blocker, reduces the phagocytic function of activated rat microglia by 40%. To further confirm a role of Nav1.6 in microglial activation, we examined the phagocytic capacity of microglia from med mice, which lack Nav1.6 channels, and show a 65% reduction in phagocytic capacity compared with microglia from wildtype mice. Our findings indicate that sodium channels are important for activation and phagocytosis of microglia and macrophages in EAE and MS and suggest that, in addition to a direct neuroprotective effect on axons, sodium channel blockade may ameliorate neuroinflammatory disorders via anti-inflammatory mechanisms.