DEVELOPMENT OF NEW OLIGODENDROCYTE-BASED MODELS TO IDENTIFY PHARMACOLOGICAL AGENTS WITH REMYELINATING ACTIVITY
Multiple Sclerosis (MS) is a chronic autoimmune disease affecting the central nervous system, characterized by neuroinflammation and demyelination. While immunomodulatory disease-modifying drugs offer relief from MS symptoms in its relapsing-remitting forms, they do not support remyelination in the progressive and most severe forms of the disease. This is because, up to date, drugs do not target the myelin-producing cells of the central nervous system: oligodendrocytes (OLs). To produce myelin and remyelinate, OLs must differentiate from oligodendrocyte precursor cells (OPCs) to their most mature form (mOLs). At intermediate stages, immature OLs (iOLs) transiently express in their cell membrane the G protein-coupled receptor 17 (GPR17). This expression is essential for their correct maturation; however, it needs to be downregulated to allow proper OL maturation. Accumulation of GPR17-positive cells has been observed in various neurodegenerative disease models, including demyelination and in the inflamed normal-appearing white matter of human MS post-mortem brains, suggesting a potential role of GPR17-positive cells in inflammation. In the first part of the present thesis, we aimed at establishing an inflammatory model of OL differentiation impairment that is representative of MS and targeting GPR17 in this model. We developed an in vitro model of inflammation to rat primary OPCs exposing them to a cocktail of pro-inflammatory cytokines commonly found in MS brain lesions: IFN-γ, TNF-α, and IL-1β (CTK). We observed that subtoxic concentrations of CTK (2 ng/mL) significantly hindered OPC differentiation, reducing the number of myelin basic protein (MBP)-positive cells and increasing the gene expression of GPR17 and chemokines associated with infiltration of leukocytes into the MS brain (CCL-2, CCL-5 and CXCL-10). In this model, we evaluated whether different GPR17 ligands could facilitate OL maturation. We demonstrated that, after a single 6-hour pretreatment with CTK, the selective GPR17 agonist galinex promoted OL maturation increasing the number of MBP-positive cells, without affecting OL inflammation genes. Conversely, montelukast, a GPR17 antagonist and known anti-inflammatory drug, together with a novel a putative partial agonist PCD-2 could not significantly counteract the effects induced by CTK. Given that myelination is a tightly regulated phenomenon that relies on both OL-intrinsic and extrinsic features, in our second strategy, we aimed at establishing oligodendrocyte-enriched human brain organoids to study the effects of repurposed remyelinating drugs on different OLs subclusters using single-cell RNA sequencing. For this, fibroblasts of a healthy donor were dedifferentiated to human induced pluripotent stem cells (hiPS) and then to neural precursor cells (NPCs). NPCs were transfected to express the Sox10 transcription factor and kept under constant agitation to obtain organoids. We treated organoids with MTK and lovastatin (LOVA) for four weeks and performed single-cell RNA sequencing (scRNA-seq). Control-, MTK- and LOVA-treated organoids presented a similar number of neurons, astrocytes, NPCs and OLs, these last in turn distributed into OPCs, iOLs and mOLs. The comparison between differentially expressed genes (DEGS) induced by both MTK and LOVA treatment allowed the identification of 377 common genes when considering the entire organoid, and of 64 common exclusive genes belonging to iOL cluster. Functional enrichment analysis showed that MTK and LOVA iOLs datasets presented common enriched terms that included the Wnt and protein tyrosine kinase pathways and glial differentiation, indicating that the drugs share possible protective mechanisms of action in this cluster. Overall, the present study sheds light on the potential of the GPR17 agonist galinex to support OL maturation under inflammation and identifies pathways of interest to give rise to novel compounds that could offer new promising therapeutic means for MS.
Networked Digital Library of Theses and Dissertations