The ADGRB1 receptor (BAI1): A key player in phagocytosis, brain development, and neurological disease Restricted; Files Only

Abstract

The adhesion G protein-coupled receptor ADGRB1 (also known as BAI1) plays an important role in suppressing angiogenesis, mediating phagocytosis, and acting as a tumor suppressor. Previous research demonstrated in vitro and in vivo knockdown of ADGRB1 leads to immature dendrite formation while overexpression leads to dendrite retraction. ADGRB1 also interacts with autism-relevant proteins such as Neuroligin 1 and IRSp53. However, little is known about the relationship between altered ADGRB1 function and clinically relevant neurological phenotypes. Therefore, in the first part of my thesis research, I evaluated the effect of reduced full-length ADGRB1 expression on behavior, seizure susceptibility, and brain morphology in homozygous (Adgrb1^exon2-/-) mice, heterozygous (Adgrb1^exon2+/-) mice, and wild-type (WT) littermates. I found that adult Adgrb1^exon2-/- mice show cognitive and social deficits, and increased vulnerability to induced seizures. Adult Adgrb1^exon2−/− mice also had significantly lower brain to body weight ratio and reduced neuron density in the hippocampus. Finally, a higher level of apoptotic cells in the hippocampus of Adgrb1^exon2-/- mice at postnatal day 1 and 7 was observed. Since ADGRB1 is a phagocytic receptor, and Adgrb1 is highly expressed in astrocytes but not in microglia, I hypothesized that astrocytes lacking full-length ADGRB1 would have reduced phagocytic ability, which in turn might contribute to the greater number of apoptotic cells observed in the hippocampus of P1 and P7 Adgrb1^exon2-/- mice. Thus, in the second part of my thesis research, I examined the phagocytic function of ADGRB1 in astrocytes and microglia. Cultured astrocytes from Adgrb1^exon2-/- mice exhibited less phagocytic capacity when compared to similarly prepared astrocytes from WT littermates. In contrast, there was no significant difference in the phagocytic capacity of cultured microglia from Adgrb1^exon2-/- mice and WT littermates. Immunostaining of pre synaptic and post synaptic markers also revealed a significantly higher volume of excitatory synapses in brain sections from adult Adgrb1^exon2-/- mutants. These results demonstrate that Adgrb1^exon2-/- mutants have reduced astrocyte-mediated phagocytosis which may result in higher levels of uncleared apoptotic cells and unwanted synapses.

Table of Contents

Table of Contents

CHAPTER 1: Introduction. 1

1.1 Overview.. 2

1.2 What are G protein-coupled receptors?. 2

1.3 Adhesion GPCRs. 5

1.4 Brain-specific angiogenesis inhibitor Family (ADGRB) 6

1.5 Brain-specific angiogenesis inhibitor 1 (ADGRB1) 8

1.5.1 Structure and signaling pathway of ADGRB1. 8

1.5.2 Function of the ADGRB1. 11

1.5.3 Mouse model lacking full-length ADGRB1. 13

1.6 Phagocytosis in the central nervous system (CNS) 14

1.6.1 Microglial mediated phagocytosis. 15

1.6.2 Astrocytic mediated phagocytosis. 16

1.7 Summary. 17

Chapter 2: Mice lacking full length Adgrb1 (Bai1) exhibit social deficits, increased seizure susceptibility, and altered brain development 19

2.1 Abstract 20

2.2 Introduction. 21

2.3 Material and methods. 22

2.3.1 Animals. 22

2.3.2 Survival and body growth curve analysis. 23

2.3.3 Brain weight assessment 23

2.3.4 Immunofluorescence staining and imaging. 23

2.3.5 Western Blot 24

2.3.6 Behavioral analysis. 25

2.3.7 Seizure induction. 27

2.3.8 Statistical analysis. 28

2.4 Result 29

2.4.1 Adgrb1^–/– mice show delayed growth and reduced brain weights. 29

2.4.2 2-3 month old Adgrb1^–/– mice exhibit reduced neuron density in the dentate gyrus and CA1. 33

2.4.3 Adgrb1^–/– mice exhibit higher levels of cleaved caspase-3-positive cells in the hippocampal CA1 and primary somatosensory cortex during early postnatal development 35

2.4.4 Adgrb1^-/- mice exhibit deficits in social behavior and learning and memory. 36

2.4.5 Adgrb1^-/- mice are susceptible to induced seizures. 39

2.5 Discussion. 41

2.6 Acknowledgement 43

2.7 Funding sources. 43

Chapter 3: ADGRB1 contributes to astrocytic phagocytosis of excitatory synapses. 45

3.1 Introduction and Background. 46

3.2 Materials and Methods. 47

3.2.1 Animals. 47

3.2.2 Immunostaining and imaging and analysis. 48

3.2.3 Primary astrocyte culture. 50

3.2.4 Primary microglia culture. 51

3.2.5 pH indicator-conjugated synaptosomes preparation. 51

3.2.6 In vitro phagocytosis uptake and analysis. 52

3.2.7 Statistical analysis. 52

3.3 Results. 53

3.3.1 P7 Adgrb1^exon2−/− mice have higher numbers of CC3+ cells and GFAP levels. 53

3.3.2 Adgrb1^exon2−/− astrocytes engulf fewer synaptosomes. 55

3.3.3 Adgrb1^exon2−/− astrocytes engulf fewer pre-synaptic elements in vivo. 57

3.3.4 Adult Adgrb1^exon2−/− mice have more excitatory but not inhibitory synapses. 58

3.4 Discussion. 60

3.5 Acknowledgment 62

3.6 Funding source. 63

Chapter 4. Conclusion and future directions. 64

4.1 Summary of the dissertation research. 65

4.2 Impact of my research on the field. 66

4.2.1 The relationship between ADGRB1 and ASD.. 66

4.2.2 Potential mechanisms by which ADGRB1 modulates learning and memory, social behavior, and seizure susceptibility. 67

4.2.3 Astrocytes vs microglia mediated phagocytosis. 68

4.2.4 Different behavioral manifestation between mouse lacking full-length ADGRB1 and mouse lacking full-length ADGRB3. 69

4.3 Future directions. 70

4.3.1 Positive modulation of ADGRB1-mediated signaling in neurological disorders. 70

4.3.2 The physiological role of ADGRB1 isoforms. 72

4.3.3 Using Adgrb1 floxed mice to further examine the role of ADGRB1 in astrocytes and neurons 73

4.3.4 The contribution of G-protein signaling in ADGRB1 mediated phagocytosis. 74

4.3.5 Are other functions altered in astrocytes lacking full-length ADGRB1?. 75

4.3.6 Elucidate the role of ADGRB1 in oligodendrocyte precursor cells. 76

4.4 Conclusion. 78

Reference. 79

Appendix A. Generation and initial characterization of mice lacking full length BAI3 (ADGRB3) expression 103

Abstract 104

A1.1 Introduction and Background. 105

A1.2 Materials and Methods. 106

A1.2.1 Animals. 106

A1.2.2 Western blot 107

A1.2.3 Brain and body weight assessment 108

A1.2.4 Behavior assessments. 108

A1.2.5 Seizure induction. 111

A1.2.6 Statistical analysis. 111

A1.3 Results. 112

A1.3.1 Generation and characterization of Adgrb3^∆7/∆7 mice. 112

A1.3.2 Adgrb3^∆7/∆7 mice exhibit reduced brain and body weight 114

A1.3.3 Adgrb3^∆7/∆7 mice exhibit deficits in social discrimination. 115

A1.3.4 Male Adgrb3^∆7/∆7 mice show a modest increase in susceptibility to flurothyl-induced seizures 118

A1.4 Discussion. 120

Appendix B: Supporting data for Chapter 2. 123

Appendix C: Supporting data for Chapter 3. 127

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Molecular Biology, Neuroscience
Keyword	Epilepsy Autism G protein-coupled receptor
Committee Chair / Thesis Advisor	Andrew Escayg, Emory University
Committee Members	Nigel Pedersen, Emory University Stephen Traynelis, Emory University Steven Sloan, Emory University Randy Hall, Emory University

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