In vivo identification of small molecules mediating Gpr126/Adgrg6 signaling during Schwann cell development

Abstract

Gpr126/Adgrg6, an adhesion family G protein-coupled receptor (aGPCR), is required for the development of myelinating Schwann cells in the peripheral nervous system. Myelin supports and insulates vertebrate axons to permit rapid signal propagation throughout the nervous system. In mammals and zebrafish, mutations in Gpr126 arrest Schwann cells at early developmental stages. We exploited the optical and pharmacological tractability of larval zebrafish to uncover drugs that mediate myelination by activating Gpr126 or functioning in parallel. Using a fluorescent marker of mature myelinating glia (Tg[mbp:EGFP-CAAX]), we screened hypomorphic gpr126 mutant larvae for restoration of myelin basic protein (mbp) expression along peripheral nerves following small molecule treatment. Our screens identified five compounds sufficient to promote mbp expression in gpr126 hypomorphs. Using an allelic series of gpr126 mutants, we parsed the ability of small molecules to restore mbp, suggesting differences in drug efficacy dependent on Schwann cell developmental state. Finally, we identify apomorphine hydrochloride as a direct small molecule activator of Gpr126 using combined in vivo/in vitro assays and show that aporphine class compounds promote Schwann cell development in vivo. Our results demonstrate the utility of in vivo screening for aGPCR modulators and identify small molecules that interact with the gpr126-mediated myelination program.

Keywords: Gpr126/Adgrg6; Schwann cells; adhesion GPCR; myelin; zebrafish.

Figure 1.. A small molecule suppressor screen in gpr126 hypomorphs for compounds that promote Schwann cell differentiation

(A) Schematic of zebrafish Gpr126 protein and mutant alleles. (B) Schematic of zebrafish Schwann cell development (green) around axons (gray) in cross-section. Radial sorting begins around 36 hours post-fertilization (hpf); wrapping is observed by 60 hpf. (C) Schematic of 5-6 days post-fertilization (dpf) larval zebrafish with central nervous system (CNS) myelin (blue) and myelinated nerves in the peripheral nervous system (PNS) (green). PLLn = posterior lateral line nerve. Boxed region shown in panels D-E. (D-E) Tg(mbp:EGFP-CAAX) expression (henceforth mbp:gfp) in wild-type (WT) and gpr126^st63 larvae at 5 dpf. Brackets denote spinal cord, arrows indicate PLLn, arrowheads mark emerging motor axons. Note strong mbp:gfp expression in the PLLn of WT (D-D’) but reduced expression in gpr126^st63 PLLn (E-E’). (F) Logic of gpr126^st63 suppressor screen. (G) Workflow for primary small molecule screening of gpr126^st63; mbp:gfp larvae.

Figure 2.. In vivo small molecule screening of gpr126 hypomorphs reveals five suppressor compounds

(A) mbp:gfp expression in control and (B) 25 μM forskolin (FSK) pulsed gpr126^st63 5 dpf larvae. Brackets denote spinal cord, arrows indicate PLLn, dotted lines indicate melanocytes obscuring PLLn. (C) Quantification of mbp:gfp as a percentage of larvae with each mbp PLLn phenotype at 5-6 dpf. *** p<0.001, Fisher’s Exact Test, “non-st63-like” vs “st63-like.” (D) Transformation of data from panel C. Dots indicate individual larva and are jittered to show all samples. Bars indicate mean ± SD. *** p<0.001, Student’s t-test. (E) Quantification of mbp:gfp(+) motor axons at 5-6 dpf. NS = no significant difference. (F) PLLn score and mbp:gfp(+) motor axons for 1462 Pharmakon compounds. Each square = one compound. Dots are jittered and transparent to show all samples. (G) Workflow of screens for gpr126^st63 suppressor compounds. (H) Rolipram restores PLLn mbp:gfp but not (I) motor axons in gpr126^st63. * p<0.05, Fisher’s Exact Test, “non-st63-like” vs “st63-like”.

Figure 3.. An allelic series of gpr126 parses direct versus indirect compound function.

(A) Molecular structure for screen hits (PubChem). (B) Schematic of gpr126 alleles, predicted proteins, phenotypes, and predicted drug functions based on mbp restoration. (C-U) Whole-mount in situ hybridization for mbp in wild-type (WT) or gpr126. Black arrows indicate PLLn. Fraction of gpr126 larvae with increased mbp is noted in upper right of each image. (C-G) mbp expression in control larvae. Note absence of PLLn mbp in gpr126^stl47, gpr126^st49, and gpr126^stl215 (D-F) and reduction in gpr126^st63 (G). (H-L) PLLn mbp expression is partially restored in gpr126^st49 (J), gpr126^stl215 (K), and gpr126^st63 (L) in 10 μM undecylenic acid. (M-Q) PLLn mbp is weakly increased in one larva for gpr126^st49 (O) and gpr126^stl215 (P) but strongly increased in gpr126^st63 (Q) with 10 μM naloxone. (R-U) mbp PLLn expression is increased across all alleles in 5 μM telmisartan-treated larvae.

Figure 4.. Apomorphine hydrochloride suppresses gpr126 hypomorphic phenotype at high doses.

(A-C, G-J) Whole-mount in situ hybridization for mbp in PLLn of gpr126^st63 (A-C) or gpr126^stl215 (G-J) with control, apomorphine, or forskolin (FSK) treatment at 5 dpf. Black arrows indicate PLLn. (D, K) Quantification expressed as a percentage of larvae with each mbp PLLn phenotype at 5 dpf. * p<0.05, *** p<0.001, Fisher’s Exact Test, “wild-type” vs. “mutant” (gpr126^st63 or gpr126^stl215) categories for treated vs. control. (E-F) Survival curve following 24-hour treatment with indicated concentrations of apomorphine. Dots indicate mean ± SD % alive across technical replicates. *** p<0.001, 0 vs.100 μM apomorphine. Sample numbers in Detailed Methods.

Figure 5.. Apomorphine hydrochloride is a direct activating ligand for Gpr126.

(A-B) COS-7 cells were transiently transfected with empty vector, WT and mutant (Δ6bp lesion as in gpr126^stl215) zebrafish gpr126 plasmid, and cAMP accumulation was measured after stimulation with given substances at 100 μM. Results are given as x-folds over vector control (vc: 1.0 ± 1.1 nM) (A) and vector control with 0.1 % DMSO (basal vc: 2.2 ± 3.1 nM) (B) for water and DMSO soluble substances, respectively. Bars show means ± SEM of three independent assays each performed in triplicates. * p<0.05, one-way ANOVA with Dunn’s multiple testing for each substance induced activation vs. basal levels. (C-D) Concentration-response curve of apomorphine (EC₅₀ WT: 21.5 μM Δ6bp mutant: 33.4 μM) (C) and codeine (no dose response detectable) (D). Results are given as x-folds over vector control (vc: 2.9 ± 0.6 nM) of four independent assays each performed in triplicates. Insets show structure of each molecule (PubChem).

Figure 6.. Glaucine suppresses gpr126^st63 hypomorphic phenotype likely by direct interaction with Gpr126.

(A) Structures of aporphine compound series. Core scaffold highlighted in blue. (B) Toxicity screening of aporphines from 47-54 hpf. Survival was assessed at 54 hpf. All compounds were at 50 μM unless otherwise noted. Control, 1% DMSO. n≥10 in all cases. (C-G, I-K) PLLn mbp:gfp expression in 5 dpf gpr126^st63 (C-G) or gpr126^st49 (I-K) following 50 μM compound treatment at 47-54 hpf. Indicated region is the most anterior portion of the PLLn. Scale bar, 25 μm. Control, 1% DMSO. Note increased PLLn mbp:gfp expression in gpr126^st63 treated with glaucine (E) compared to control (C) but absence of mbp:gfp in glaucine-treated gpr126^st49 (J). (H) Quantification of PLLn mbp expression within ROI of gpr126^st63 larvae in C-G. Bars indicate means ± SD. ****p<0.0001, *p<0.05, one-way ANOVA with Dunnett’s multiple testing for each compound against control.