A humanized zebrafish screening platform to identify GPR17 inhibitors for the treatment of multiple sclerosis

Abstract

Multiple sclerosis (MS), a demyelinating disease of the central nervous system (CNS) with dev-astating symptoms, is characterized by the progressive destruction of myelin and myelinating oligodendrocytes (Ol). Currently, there is still an unmet therapeutic need to promote remye-lination to treat MS. <br /> The restoration of myelin sheaths requires the invasion of oligodendrocyte precursor cells (OPC) into demyelinated lesions and their differentiation into mature myelinating Ols. Howev-er, despite the large numbers of OPC in demyelinated lesions of MS patients, remyelination fails. This indicates insufficient Ol differentiation due to either the absence of pro-myelinating signals or the presence of myelination inhibitors in the MS lesion. One such myelination inhibi-tor is the GPR17 gene, which codes for an orphan G protein-coupled receptor (GPCR) that has attracted particular attention as oligodendroglial maturation inhibitor in mice. In humans, GPR17 is also disease-relevant: it is highly abundant within active white matter plaques of MS patients. Moreover, absence of GPR17 promotes remyelination in a murine autoimmune model of MS, proposing inhibitors of GPR17 as a promising therapy to promote remyelination in pa-tients with MS. <br /> To develop novel therapeutic approaches, animal experiments are still considered the gold standard, because in vivo experiments allow examination of both therapeutic and potential adverse effects of drugs on the whole organism. Experimental drugs that function across ani-mal species might be expected to show superior efficacy in humans, and, therefore, eventually promote remyelination in MS. Unfortunately, drugs emerging from preclinical studies in animal MS models have a poor record of success in human clinical trials, highlighting the need of “hu-manized” in vivo models. <br /> In search for improved animal models, zebrafish (Danio rerio) has emerged as a popular verte-brate model. It is a widely-used organism for studying developmental processes and for drug testing. Especially, zebrafish larvae have become a powerful tool for the in vivo study of Ol bi-ology and (re-)myelination, because of its high genetical and experimental versatility and its transparency for in vivo imaging. <br /> Therefore, this study focusses on investigating the functional role of Gpr17 in zebrafish in or-der to establish a “humanized” zebrafish screening platform for the identification of GPR17 inhibitors as a potential therapy for MS. We found that gpr17 mRNA is expressed in OPCs, pre-Ols but not in mature Ols in zebrafish. Consistent with data obtained in mice, we deciphered the functional role of Gpr17 to be an Ol differentiation inhibitor in zebrafish. Furthermore, “hu-manized” zebrafish lines expressing the human GPR17 or a chimeric GPR17, containing the ligand binding domain of human GPR17 and the intracellular loops of zebrafish Gpr17, were generated. We demonstrated that both the human and the chimeric GPR17 receptors are func-tional in zebrafish and therefore provide a tool to perform drug screenings for antagonists of human GPR17 in a humanized zebrafish model. To facilitate the search of inhibitors of human GPR17 we also developed an automated screening system using the EnSightTM multimode plate reader.