Regulation of Myelin Proteins and Protection Against Demyelination by GPR37 Signaling Open Access
Smith, Brilee Michelle (2017)
Abstract
The G protein-coupled receptor (GPCR) superfamily is the largest and most diverse transmembrane receptor family in vertebrates. For this reason, GPCR signaling is involved in numerous biological processes, and GPCRs are major pharmaceutical targets. GPR37 is an orphan GPCR 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. To develop tools to manipulate GPR37 signaling in order to better understand the receptor's physiological functions, we sought to identify compounds and interacting partners that modulate GPR37 signaling. A high-throughput screen of 16,000 compounds revealed that octoclothepin maleate is a nonspecific inhibitor of constitutive GPR37 signaling. The GPR37-interacting proteins regenerating islet-derived family member (REG)2 and REG4 were also found to inhibit GPR37 signaling. The findings presented in this dissertation 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.
Table of Contents
CHATPER I: Introduction...................................................................................................1
1.1Background and history of the study of GPCRs.............................................................2
1.2 GPCR Classification......................................................................................................3
1.3 GPCR Signaling.............................................................................................................6
1.4 Clinical relevance of GPCRs.......................................................................................15
1.5 GPR37 and GPR37L1..................................................................................................17
1.6 Oligodendroglia and myelination................................................................................26
1.7 GPCRs in myelin health and disease...........................................................................37
1.8 Aim of dissertation research........................................................................................39
CHAPTER II: GPR37 regulates expression of the myelin-associated glycoprotein MAG..................................................................................................................................40
2.1 Abstract........................................................................................................................41
2.2 Introduction..................................................................................................................42
2.3 Experimental procedures.............................................................................................44
2.4 Results..........................................................................................................................50
2.4.1 Mag protein expression, but not Mag mRNA expression, is decreased in brains of Gpr37-/- mice.....................................................................................................................50
2.4.2 MAG interacts with GPR37......................................................................................59
2.5 Discussion....................................................................................................................64
CHAPTER III: Loss of Gpr37 results in increased susceptibility to cuprizone-induced demyelination.....................................................................................................................67
3.1 Abstract........................................................................................................................68
3.2 Introduction..................................................................................................................69
3.3 Experimental procedures.............................................................................................71
3.4 Results..........................................................................................................................75
3.4.1 Loss of Gpr37 increases susceptibility to cuprizone-induced demyelination...........75
3.4.2 Loss of Gpr37 does not affect density of OPCs or mature oligodendrocytes during cuprizone-induced demyelination......................................................................................86
3.5 Discussion....................................................................................................................96
CHAPTER IV: Identification of modulators of GPR37 signaling..................................101
4.1 Abstract......................................................................................................................102
4.2 Introduction................................................................................................................103
4.3 Experimental procedures...........................................................................................106
4.4 Results........................................................................................................................110
4.4.1 High throughput screening for small molecule modulators of GPR37 signaling...110
4.4.2 Effect of interacting partners of GPR37 signaling..................................................127
4.5 Discussion..................................................................................................................137
CHAPTER V: Further discussion and future directions..................................................140
References........................................................................................................................163
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