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ScRNAseq of human oligodendrocytes in primary culture exposed to glutamate vs. vehicle

ABSTRACT: T helper (Th)17 cells are considered to contribute to inflammatory mechanisms in diseases such as multiple sclerosis (MS). However, the discussion persists regarding their true role in patients. Here, we visualized central nervous system (CNS) inflammatory processes in models of MS live in vivo and in MS brains and discovered that CNS-infiltrating Th17 cells form prolonged stable contact with oligodendrocytes. Strikingly, compared to Th2 cells, direct contact with Th17 worsened experimental demyelination, caused damage to human oligodendrocyte processes and increased cell death. Importantly, we found that in comparison to Th2 cells, both human and murine Th17 cells express higher levels of the integrin CD29, which is linked to glutamate release pathways. Of note, contact of human Th17 cells with oligodendrocytes triggered release of glutamate. As we found that Th17-polarized cells release glutamate in contact with oligodendrocytic cells, we then assessed the impact of glutamate itself on human oligodendrocytes in primary culture. We found that 12-24h application of glutamate - but not pro-inflammatory cytokines TNFα or IFN-γ - strongly decreased the area and complexity of human oligodendrocyte processes in vitro without affecting oligodendrocyte survival. To further investigate the impact of glutamate on human oligodendrocytes we used single cell RNA sequencing (scRNAseq) of the human oligodendrocytes in primary culture, unstimulated (vehicle) and stimulated with glutamate. Following exposure to glutamate we observed induced cell stress and changes in biosynthesis of cholesterol and lipids in mature human oligodendrocytes. Finally, exposure to glutamate decreased myelination whereas blockade of CD29 preserved oligodendrocyte processes from Th17-mediated injury. Our data provide first evidence for direct and deleterious attack of Th17 cells on the myelin compartment and show the potential for new therapeutic opportunities in MS.

ORGANISM(S): Homo sapiens

PROVIDER: GSE180670 | GEO | 2021/07/25

REPOSITORIES: GEO

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Project description:Multiple sclerosis is a chronic neuroinflammatory disorder characterized by demyelination, oligodendrocyte damage/loss and neuroaxonal injury in the context of immune cell infiltration in the central nervous system. No neuroprotective therapy is available to promote the survival of oligodendrocytes and protect their myelin processes in immune-mediated demyelinating diseases. Pro-inflammatory CD4 T helper (Th)17 cells can directly interact with oligodendrocytes in multiple sclerosis and its animal model, causing injury to myelinating processes and cell death through direct contact. However, the molecular mechanisms underlying the close contact and subsequent detrimental interaction of Th17 cells with oligodendrocytes remain unclear. In this study we conducted single cell RNA sequencing of human primary oligodendrocytes cultured alone, in contact or separated by an insert with primary Th17 polarized T cells and identified several cell adhesion molecules that may modulate T cell contact mediated damage of oligodendrocytes. Furthermore, we used flow cytometry and immunofluorescence studies on central nervous system tissue from multiple sclerosis subjects, its animal model and controls to characterize the expression of cell adhesion molecules by mature oligodendrocytes. We found that a significant proportion of human and murine mature oligodendrocytes express melanoma cell adhesion molecule and activated leukocyte cell adhesion molecule in multiple sclerosis, EAE and controls although their regulation differ between human and mouse. We observed that exposure to pro-inflammatory cytokines or to human activated T cells are associated with a marked downregulation of the expression of melanoma cell adhesion molecule but not activated leukocyte cell adhesion molecule at the surface of human primary oligodendrocytes. Furthermore, we used in vitro live-imaging, immunofluorescence, and flow cytometry to determine the contribution of these molecules to Th17-polarized cell adhesion and cytotoxicity towards human oligodendrocytes. Silencing and blocking activated leukocyte cell adhesion molecule but not melanoma cell adhesion molecule limited prolonged interactions between oligodendrocytes and Th17-polarized cells, resulting in decreased adhesion of Th17-polarized cells to oligodendrocytes and conferred significant protection of oligodendrocytic processes.

Project description:Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients. RNA sequencing of oligodendrocyte progenitor cells treated with vehicle, miconazole or clobetasol for 0, 2, 6, or 12 hours. Cells were plated 1.5 hours prior to addition of drug.

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ScRNAseq of human oligodendrocytes in primary culture exposed to glutamate vs. vehicle

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