Adgrg6 drug discovery using the zebrafish model

Schwann cells are the principal myelinating glial cells of the Peripheral Nervous System (PNS) that insulate neuronal axons with a lipid-rich myelin sheath. Defects of the myelin sheath can impair nerve conduction and are associated with various developmental and demyelinating diseases, including those involving Schwann cell pathobiology that lead to weakness, paralysis and death in severe cases. Lethal Congenital Contracture Syndrome 9 (LCCS9) is an example in which myelination of the PNS is defective as a result of a missense mutation in ADGRG6, which encodes the adhesion G protein-coupled receptor G6 (ADGRG6, formerly GPR126) with conserved roles in Schwann cell maturation. adgrg6 is expressed in zebrafish and has conserved regulatory roles in myelination of the PNS and additional roles in semicircular canal formation in the inner ear. As part of an ongoing drug-screening project, I set out to identify small molecules that are potent and specific modulators of the Adgrg6 signalling pathway, using zebrafish larvae as a whole-animal screening platform due to their amenability for high-throughput drug screening and gene expression analysis by whole-mount in situ hybridisation. In the current work, I first characterised the peripheral myelination phenotype in hypomorphic adgrg6tb233c mutant larvae, before optimising pharmacological conditions for a screening assay using myelin basic protein (mbp) mRNA expression in the PNS as a transcriptional readout of Adgrg6 pathway activity. We previously identified candidate pathway modulators from the Spectrum and Tocris compound libraries, using otic versican b (vcanb) mRNA expression in a primary screen for phenotypic rescue of adgrg6 hypomorphic mutants, before testing hits on a counter-screening mbp assay. I have similarly screened the Sigma LOPAC library of 1280 pharmacologically active compounds. This collection contains 685 compounds not represented in our previous screens, of which 275 are structurally diverse from any compounds we have previously tested. The LOPAC screen has identified 42 hit compounds that can downregulate vcanb in otic tissue, 17 of which also restore mbp expression around the posterior lateral line ganglion of adgrg6 hypomorphic mutant larvae. Hit compounds include classes of molecules identified previously, including dihydropyridines, together with some new candidate modulators of the Adgrg6 signalling pathway; those that are ineffective at rescuing the otic phenotype in a truncating mutant allele are likely to interact directly with the Adgrg6 receptor. Preliminary cAMP assays in HEK293 cells failed to illustrate potential agonistic effects of candidate compounds including, ivermectin and ebastine from the LOPAC library. However, testing ADGRG1 antagonists, including dihydromunduletone (DHM), revealed potential antagonism towards the zebrafish Adgrg6 receptor. Moreover, preliminary work in zebrafish revealed that wild-type larvae could somewhat phenocopy adgrg6 hypomorphic mutants following DHM injection into the larval ear. Further work is necessary to confirm observations in this study; however, the discovery of Adgrg6 compound modulators will provide useful tools to modulate the receptor’s pathway activity, with the potential to give additional insights into its underlying mechanism of action. Compounds could also hold potential for therapeutic use against demyelinating or ADGRG6-linked human diseases.