Mice lacking Gpr37 exhibit decreased expression of the myelin-associated glycoprotein MAG and increased susceptibility to demyelination

Abstract

GPR37 is an orphan G protein-coupled receptor that is predominantly expressed in the brain and found at particularly high levels in oligodendrocytes. GPR37 has been shown to exert effects on oligodendrocyte differentiation and myelination during development, but the molecular basis of these actions is incompletely understood and moreover nothing is known about the potential role(s) of this receptor under demyelinating conditions. To shed light on the fundamental biology of GPR37, we performed proteomic studies comparing protein expression levels in the brains of mice lacking GPR37 and its close relative GPR37-like 1 (GPR37L1). These studies revealed that one of the proteins most sharply decreased in the brains of Gpr37/Gpr37L1 double knockout mice is the myelin-associated glycoprotein MAG. Follow-up Western blot studies confirmed this finding and demonstrated that genetic deletion of Gpr37, but not Gpr37L1, results in strikingly decreased brain expression of MAG. Further in vitro studies demonstrated that GPR37 and MAG form a complex when expressed together in cells. As loss of MAG has previously been shown to result in increased susceptibility to brain insults, we additionally assessed Gpr37-knockout (Gpr37^-/-) vs. wild-type mice in the cuprizone model of demyelination. These studies revealed that Gpr37^-/- mice exhibit dramatically increased loss of myelin in response to cuprizone, yet do not show any increased loss of oligodendrocyte precursor cells or mature oligodendrocytes. These findings reveal that loss of GPR37 alters oligodendrocyte physiology and increases susceptibility to demyelination, indicating that GPR37 could be a potential drug target for the treatment of demyelinating diseases such as multiple sclerosis.

Keywords: GPCR; cuprizone; demyelination; multiple sclerosis; myelin; remyelination.

Figure 1. Expression of MAG protein, but not MAG mRNA, is decreased with loss of GPR37

Representative Western blot images (A) and quantification (B) for total MAG protein in whole brain tissue samples from WT and Gpr37^−/− mice showed significantly decreased MAG protein expression in Gpr37^−/− and DKO mice, but not Gpr37L1^−/− mice (n=8 per genotype, data analyzed using one-way ANOVA *p<0.01, **p<0.001). Molecular mass markers (in kDa) are shown on the left side of the blots. C) Relative Mag mRNA expression was not significantly different between whole brain samples from WT and Gpr37^−/− mice, as assessed via qPCR

Figure 2. Expression of other oligodendrocyte and myelin proteins is unaffected by loss of GPR37

Representative Western blot images (A) and quantification of expression of the myelin- associated proteins Mbp (B), Mog (C), and Gstπ(D) in whole brain tissue samples from WT and Gpr37^−/− mice revealed no significant differences in protein expression. All protein levels were normalized to actin expression (n=8 per genotype). Molecular mass markers (in kDa) are shown on the left side of each immunoblot (IB).

Figure 3. GPR37 coimmunoprecipitates with MAG, but does not increase MAG expression upon co-transfection in vitro

A) GFP-tagged GPR37, but not Flag-tagged M1 muscarinic acetylcholine receptor, coimmunoprecipitates with MAG. Molecular mass markers are shown (in kDa) on the left side of the blots. Both GFP-GPR37 and Flag-M1 exhibit higher-order oligomers in addition to bands corresponding to monomeric receptors. B-C) Cotransfection of GFP-tagged GPR37 with MAG did not increase MAG expression in HEK-293 cells. All protein levels were normalized to actin expression (n=7 per genotype). IP=immunoprecipitation, IB=immunoblot, GFP37=GFP-tagged GPR37, M1=Flag-tagged M1 muscarinic acetylcholine receptor, EV=Empty vector.

Figure 4. Loss of GPR37 increases susceptibility to demyelination

Representative images (A) and quantification (B) of luxol fast blue (LFB)-stained corpus callosum from cuprizone-treated mice. Gpr37^−/− mice displayed significantly more demyelination than wild-type (WT) mice at weeks 4, 6, and 9 of cuprizone administration. LFB analysis of myelination was done by objective, blinded scoring on a scale of 0–4: 0=completely demyelinated, 1=25% myelinated, 2=50% myelinated, 3=75% myelinated, 4=100% myelinated. Data analyzed using two-way ANOVA with Sidak post-hoc analysis **p<0.01, ***p<0.001, ****p<0.0001

Figure 5. Loss of GPR37 does not affect numbers of OPCs

Representative images (A) and quantification (B) of NG2+ oligodendrocyte precursor cells (OPCs) in the corpus callosum of mice treated with cuprizone. No significant difference between wild-type (WT) and Gpr37^−/− mice was observed at any time point.

Figure 6. Loss of GPR37 does not affect numbers of mature oligodendrocytes

Representative images (A) and quantification (B) of Gstπ+ mature oligodendrocytes in the corpus callosum of mice treated with cuprizone. No significant difference between wild-type (WT) and Gpr37^−/− mice was observed at any time point.