Author Of 4 Presentations
P0776 - Dual threats of CSPGs in multiple sclerosis through inhibiting oligodendrocyte repair and polarizing Th17 cells (ID 1167)
Abstract
Background
In multiple sclerosis (MS), oligodendrocytes and myelin are damaged by immune responses. Remyelination failure contributes to the axonal loss and progression of disability. The failed repair process could be the consequence of ongoing toxic neuroinflammation and to inhibitors in lesions. The extracellular matrix (ECM) molecules, including members of the chondroitin sulfate proteoglycan (CSPG) family, contribute to the altered microenvironment of MS lesions. CSPGs, particularly versican V1, have been described to directly inhibit the differentiation of oligodendrocyte precursor cells (OPCs) and to prevent remyelination.
Objectives
Herein, we examined ECM members for their expression in lesions of MS and experimental autoimmune encephalomyelitis (EAE), a model of MS, and addressed whether these ECM molecules alter immune responses that then affect tissue repair in MS lesions.
Methods
EAE was induced with MOG peptide 35-55 in CFA and spinal cord tissues were evaluated.Immunofluorescence staining and confocal microscopy were employed to determine which members of ECM are altered in EAE lesions.To assess the effect of ECM molecules on T cells, isolated T cells from 2D2 mice were cultured on ECM coated wells and were then activated with MOG-loaded dendritic cells and polarized to Th17 subset. The frequency of Th17 was determined using flow cytometry 5 days later.
Results
we found increased levels in spinal cord EAE lesions of versican V1, fibronectin, thrombospondin-1 and heparan sulfate proteoglycans (HSPGs), but not versican V2 or aggrecan. Versican V1 deposition was confirmed in active MS lesions. In culture, a mixed CSPG preparation and purified versican V1, but not other ECM molecules tested, shifted T cell differentiation toward Th17 cells, which were then inhibitory for OPC differentiation. CSPG-polarized Th17 cells also killed OPCs. To inhibit CSPG synthesis in vivo, EAE mice were injected with difluorosamine (peracetylated,4,4-difluoro-N-acetylglucosamine) starting at the peak of clinical disease severity. Difluorosamine-treated mice has reduced injury-enhanced versican content in spinal cord lesions correspondent with lower frequency of Th17 cells. To study the effect of difluorosamine on remyelination during EAE, NG2-Cre:Tau-mGFP mice were used with GFP expression localized to newly formed oligodendrocytes and myelin. We found that difluorosamine reduced levels of versican V1 in well-formed EAE lesions resulting in higher number of new oligodendrocytes and myelin sheets around and within lesions
Conclusions
These results highlight lesion-elevated CSPGs, especially versican V1, in directly inhibiting OPCs and indirectly in shaping T cell responses which then impact remyelination outcomes. We propose CSPG-lowering drugs as dual pronged repair therapeutics that directly affect OPCs, and that indirectly antagonize Th17 roles in inflammation and oligodendrocyte injury.
P0779 - Lipid handling by macrophages and microglia is impaired in the aging CNS after demyelination (ID 578)
Abstract
Background
Multiple Sclerosis (MS) is a chronic inflammatory, demyelinating neurodegenerative disease of the central nervous system (CNS). Demyelination results in an accumulation of myelin debris, which is mostly comprised of lipids, in the extracellular space, which previous studies have shown to impair remyelination. Under optimal circumstances, the debris is cleared through phagocytosis by phagocytes, such as microglia and macrophages.
Objectives
A key determinant of progression of most neurodegenerative diseases including MS is age. Studies have reported the deficient clearance of myelin debris, and increased formation and deposition of cholesterol crystals within macrophages, in the aging CNS after demyelination. This has been associated with reduced expression of cholesterol exporter ABCA1 with age. As myelin contains about 80% of lipids, we propose that the accumulation, deficient processing and shuttling of lipids or lipid droplets contribute to the impaired myelin debris clearance in an injured aging CNS.
Methods
We have used lysolecithin demyelination in the spinal cord of young (2-3 months) and aging (8-12 months) mice. Confocal and slide scanner microscopes were utilized to investigate the abundance of lipid droplets in young and aging mice. We conducted bulk RNA sequencing of laser-microdissected lesion sites at day 3 post demyelination. In culture, we have used the macrophages and microglia to assess the effect of lipids on phagocytosis of myelin debris.
Results
In bulk RNA sequencing of laser-microdissected lesion sites at day 3 post demyelination, we found that transcripts encoding the apparatus associated with lipid processing and their degradation (such as pxmp2, cat, abcd3, rab37, lcat) are greatly reduced with aging. Confocal microscopy documented the accumulation of lipid droplets within macrophage/microglia in lesions, and this is significantly higher at day 14 and 21 in aging compared to young mice. In culture, we found that extracellular cholesterol increases the engulfment of myelin debris by macrophages and microglia, leading to appearance of foamy cells. Current experiments probe the contribution of the deficient handling of myelin associated lipids in cholesterol crystal formation and how to target therapeutically the processing of intracellular lipids.
Conclusions
The proper and efficient handling of myelin lipid is imperative for making the lesion environment more conducing for repair.
P0919 - The Canadian Prospective Cohort (CanProCo) Study to Understand Progression in Multiple Sclerosis: Rationale and Baseline Characteristics (ID 1236)
Abstract
Background
Neurological disability progression occurs across the spectrum of people living with multiple sclerosis (PwMS). Currently, no treatments exist that substantially modify the course of clinical progression in MS, one of the greatest unmet needs in clinical practice. Characterizing the determinants of clinical progression is essential for the development of novel therapeutic agents and treatment approaches that target progression in PwMS.
Objectives
The overarching aim of CanProCo is to evaluate a wide spectrum of factors associated with the onset and rate of disease progression in MS, and to describe how these factors interact with one another to influence progression.
Methods
CanProCo is a prospective, observational cohort study aiming to recruit 1000 individuals with radiologically-isolated syndrome (RIS), relapsing-remitting MS (RRMS), and primary-progressive MS (PPMS) within 10-15 years of disease onset, and 50 healthy controls (HCs) from five large academic MS centers in Canada. Participants undergo detailed clinical evaluations annually. A subset of participants enrolled within 5-10 years of disease onset (n=500) also have blood, cerebrospinal fluid, and MRIs collected facilitating study of biological measures (e.g. single-cell RNA-sequencing[scRNASeq]), MRI-based microstructural assessment, participant characteristics (self-reported, performance-based, clinician-assessed, health-system based), and environmental factors as determinants contributing to the differential progression in MS.
Results
Recruitment commenced in April/May 2019 and n=536 patients have been recruited to date (RRMS=457, PPMS=35, RIS=25, HC=19). Baseline age, sex distribution, and Expanded Disability Status Scale (EDSS) scores (median, range) of each subgroup are: RRMS=38 years, 73% female, EDSS=1.5 (0-6.0); PPMS=52 years, 40% female, EDSS=4.0 (1.5-6.5); RIS=41 years, 68% female, EDSS=0 (0-3.0); HC=37 years, 63% female. Recruitment has surpassed the 50% target but has been paused due to the COVID-19 pandemic. scRNASeq on frozen blood samples has been validated.
Conclusions
Halting the progression of MS is a fundamental clinical need to improve the lives of PwMS. Achieving this requires leveraging transdisciplinary approaches to better characterize mechanisms underlying clinical progression. CanProCo is the first prospective cohort study aiming to characterize these determinants to inform the development and implementation of efficacious and effective interventions.
P0946 - CCR2+ monocytes require EMMPRIN for migration into EAE/MS brains (ID 1169)
Abstract
Background
Multiple sclerosis (MS) is a demyelinating condition of the central nervous system (CNS) mediated by a complex interplay of adaptive and innate immune cells. In MS and its model, experimental autoimmune encephalomyelitis (EAE), the receptor for CCL2 chemokine, CCR2, is critical for monocyte/macrophage migration into the CNS to exert their effector pathological functions. Recently, we showed that extracellular matrix metalloproteinase inducer (EMMPRIN), a glycoprotein present on monocyte/macrophage membrane, aids aerobic glycolysis (a pro-inflammatory metabolic program) within inflammatory macrophages by regulating the surface levels of monocarboxylate transporter (MCT-4), a lactate transporter.
Objectives
Our earlier studies found that neutralization of EMMPRIN activity by function blocking antibodies ameliorated EAE severity. Here, to understand specific functions and mechanisms of EMMPRIN on CCR2+ monocyte/macrophages, we investigated genetic deletion of EMMPRIN in CCR2+ monocytes.
Methods
Full length EMMPRIN was floxed using CRISPR/Cas9 (inserted 5’ flox in 1st exon and 3’ flox in 8th exon of EMMPRIN gene, Bsg2). These EMMPRIN ‘floxed’ mice were then crossed with CCR2CreERT2+mKate2 mice to yield CCR2CreERT2:EMMPRINfl/fl (EMMPRIN-/- CCR2+) tamoxifen inducible mice. EMMPRIN deletion was induced by injection of 2 mg tamoxifen/mouse every 48h starting at day 2 post EAE induction. The last injection was given on day 14. Some groups had resumption of tamoxifen regimen at day 36 until harvest. In vitro, bone marrow derived macrophages (BMDMs) were exposed to 5μM 4-hydroxytamoxifen for 24h before stimulating with LPS for another 24h for quantitative proteomics and qRT-PCR analysis.
Results
EMMPRIN-/- CCR2+ mice exhibited significantly delayed onset of EAE signs; disease severity was significantly reduced after clinical signs set in. EMMRIN-/- CCR2+ mice had significantly reduced number of perivascular cuffs during peak EAE (day 18), during which the spinal cord had significantly reduced CCR2+EMMPRIN+ monocyte/macrophages. These mice harbored elevated numbers of CCR2+ monocytes in lymph nodes suggesting an impairment of trafficking of EMMPRIN-/- CCR2+ monocytes into the CNS. In vitro, we observed alteration of proteome in EMMPRIN-/- macrophages, which was skewed towards gluconeogenesis metabolic program.
Conclusions
Selective EMMPRIN deletion in CCR2+ monocyte/macrophages alters metabolic program and interferes with their migration into EAE CNS. EMMPRIN on myeloid cells may constitute a therapeutic target in MS.