Protective Actions of the Brain-Expressed Receptors GPR37 and GPR37L1 in Models of Neurological Disease Open Access

Giddens, Michelle Marie (2017)

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G protein-coupled receptors (GPCRs) are essential for cellular communication. In the central nervous system, GPCRs serve as the molecular targets for many neurotransmitters, neuropeptides, and neuromodulators. Therefore, it is not surprising that GPCRs are essential for most physiological processes and their dysfunction contributes to the etiology of many disease states and disorders. Furthermore, GPCRs constitute one of the largest classes of drug targets in the body. While the functions of many GPCRs are well characterized, a subset of GPCRs remain poorly understood. These receptors have the potential to increase understanding of basic biology, and/or serve as novel therapeutic targets. Here, we present evidence that GPR37 and GPR37L1, a pair of closely related receptors that are predominantly expressed in the brain, exert protective actions in models of seizure and stroke.

My colleagues and I demonstrated that a variant in GPR37L1 is associated with a novel human progressive myoclonic epilepsy. We also found that loss of Gpr37L1 leads to increased seizure susceptibility in two seizure induction models. Additionally, we demonstrated that loss of GPR37 increases seizure susceptibility and loss of both Gpr37 and Gpr37L1 in double knockout (DKO) mice leads to an even more dramatic effect, suggesting loss of both receptors is synergistic.

In separate studies, we demonstrated that expression of GPR37 is dramatically elevated in the penumbra following ischemia, and that loss of GPR37 results in increased infarct volume in a focal cerebral ischemia model. Conversely, GPR37L1 expression was significantly reduced following ischemic insult in WT mice, but not GPR37-/- mice suggesting a compensatory relationship between the two receptors in vivo. The results of these studies also demonstrated that loss of GPR37 results in attenuated HIF1α and GFAP expression following ischemia. Finally, astrocytes cultured from Gpr37-/- mice were found to be more susceptible to an in vitro model of stroke.

The findings described in this dissertation provide evidence that GPR37 and GPR37L1 function as protective receptors in the central nervous system in seizure models and a cerebral ischemia model. These observations mark GPR37 and GPR37L1 as novel therapeutic targets for the treatment of epilepsy, stroke and other neurological disorders.

Table of Contents


CHAPTER 1: Introduction to G Protein-Coupled Receptors 1

1.1 Cell Communication: Role of GPCRs 2

1.1.1 Discovery of GPCRs 2

1.1.2 GPCR Classification 3

1.1.3 GPCR Signaling 4

1.1.4 Common GPCR Interacting Partners 11

1.2 GPCRs in Health and Disease 13

1.2.1 GPCRs as Pharmaceutical Targets 13

1.2.2 Orphan GPCRs 14

1.3 GPR37 and GPR37L 15

1.3.1 Identification and Expression 15

1.3.2 Receptor Processing 19

1.3.3 Interacting Partners 21

1.3.4 Ligand Identification 22

1.3.5 Functions of GPR37 & GPR37L1 In Vivo 25

1.3.6 Disease Associations 27

1.3.7 Protective Mechanisms 29

1.4 Dissertation Goals 29

CHAPTER 2: GPR37L1 Modulates Seizure Susceptibility: Evidence from Mouse Studies and Analyses of a Human GPR37L1 Variant 31

2.1 Rationale 32

2.2 Experimental Methods 33

2.2.1 Clinical Reports 33

2.2.2 Human Exome Sequencing 35

2.2.3 Sanger Confirmation 36

2.2.4 Cell culture 36

2.2.5 Western Blotting 36

2.2.6 Cell Surface Biotinylation 37

2.2.7 Confocal Microscopy 37

2.2.8 Phosphorylation assays 38

2.2.9 Luciferase reporter assays 39

2.2.10 cAMP Assay 40

2.2.11 Co-immunoprecipitation Assays 41

2.2.12 Degradation Experiments 41

2.2.13 Generation of Knockout Mice (KO) and Maintenance of Mouse Colony 42

2.2.14 6 Hz Seizure Induction 43

2.2.15 Flurothyl Seizure Induction 43

2.2.16 EEG Analyses 44

2.3 Results 44

2.3.1 A homozygous variant in GPR37L1 (K349N) implicates GPR37L1 in seizure susceptibility 44

2.3.2 The K349N substitution does not alter receptor expression or trafficking 49

2.3.3 Constitutive signaling by GPR37L1 is not affected by the K349N mutation 54

2.3.4 GPR37L1 is ubiquitinated in HEK293T cells, and the K349N substitution does not alter ubiquitination 65

2.3.6 Mice lacking Gpr37L1, Gpr37 or both receptors, are susceptible to 6 Hz induced seizure 72

2.3.7 Mice lacking GPR37L1, but not GPR37, are more susceptible to Flurothyl induced seizure 76

2.3.8 Mice lacking both GPR37L1 and GPR37 have spontaneous seizures 78

2.4 Summary and Discussion 80

CHAPTER 3: Loss of GPR37 Results in Increased Infarct Volume and Altered Astrocytic Response in Models of Stroke 84

3.1 Rationale 85

3.2 Experimental Methods 87

3.2.1 Generation of Knockout Mice and Maintenance of Mouse Colony 87

3.2.2 Induction of a Focal Cortical Ischemic Stroke 87

3.2.3 Infarct Volume Measurement 88

3.2.4 Western Blotting 88

3.2.5 Cell Culture 89

3.2.6 Oxygen Glucose Deprivation/Reperfusion 90

3.2.7 Cytotoxicity 90

3.3 Results 91

3.3.1 Loss of Gpr37 leads to increased infarct size in a cortical ischemic stroke model 91

3.3.2 Expression of GPR37 is dramatically increased in the penumbra following MCAO while GPR37L1 expression is significantly reduced 94

3.3.3 HIF1α expression fails to upregulate in the penumbra of Gpr37-/- mice following MCAO 97

3.3.4 Increased inflammatory markers Iba1 and GFAP in the penumbra of WT, but not Gpr37-/- mice following MCAO 99

3.3.5 Primary cultured astrocytes from Gpr37-/- mice are more susceptible to oxygen and glucose deprivation. 102

3.4 Summary and Discussion 104

CHAPTER 4: Conclusions and Future Directions 108

4.1 Summation of Dissertation Work 109

4.2 GPR37 and GPR37L1: Ligand Identification 111

4.3 Discussion and Future Directions 115

4.3.1 K349N Pathogenicity 115

4.3.2 Loss of GPR37 and/or GPR37L1 Elevates Seizure Susceptibility 117

4.3.3 Loss of Gpr37 Increases Infarct Volume 122

4.3.4 Loss of GPR37 Reduces Astrocyte Survival in an in vitro Model of Ischemia 127

4.3.5 Theoretical model of GPR37 and/or GPR37L1 contribution to seizure susceptibility and ischemic stroke 128

4.3.5 Therapeutic Potential of GPR37 and GPR37L1 133

4.4 Concluding Thoughts 134

APPENDIX I: GPR37 and GPR37L1: Protein Interaction 167

Section I.1 Rationale 168

Section I.2 Materials and Methods 169

I.2.1 Tissue Homogenization 169

I.2.2 LC-MS/MS analysis 169

I.2.3 Data Analysis 170

I.2.4 Endogenous Pulldown 171

I.2.5 Sample Digestion 172

I.2.6 Database search: 173

Section I.3 Results 174

Section I.4 Conclusions and Discussion 189

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