NPAS2 and CLOCK in the mammalian retina: their localization and roles in clock-controlled gene regulation and visual function Open Access

Hwang, Christopher Kwan (2014)

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Circadian rhythms are biological rhythms that occur on a daily basis. These rhythms are thought to be controlled by an autonomous transcriptional-translational feedback loop called the circadian clock. Traditionally, the clock has been thought to be comprised of two positive-regulator proteins (CLOCK and BMAL1) and four negative-regulator proteins. However, in certain parts of the brain, recent studies have demonstrated that NPAS2 has an overlapping role with CLOCK and led to my investigation of NPAS2's role in the retinal circadian clock. I determined that NPAS2 is expressed rhythmically in a subset of retinal ganglion cells and regulates the expression of a clock-controlled gene, adenylyl cyclase type 1 (Adcy1), selectively in the ganglion cell layer (GCL). I also discovered that Npas2-/- mice show strikingly similar reductions in the contrast sensitivity rhythm to that of Adcy1-/- and Drd4-/- mice in which contrast sensitivity has previously been shown to be disrupted. In Drd4-/- mice, I discovered that the expression of Npas2 and Adcy1 transcripts in the GCL are arrhythmic, suggesting that contrast sensitivity is modulated through a dopamine-NPAS2-adenylyl cyclase pathway in the GCL. Next I explored the role of CLOCK in the contrast sensitivity regulation and determined that contrast sensitivity in Clock-/- mice contrast sensitivity is arrhythmic. Using a luciferase reporter assay, I determined that the Adcy1 promoter is selectively activated by the NPAS2/BMAL1 heterodimer but not the CLOCK/BMAL1 heterodimer in neuronal NG108-15 cells and that the transcript expression of Npas2 and Adcy1 in the GCL of Clock-/- mice are arrhythmic, suggesting that CLOCK regulates the contrast sensitivity rhythm in part by regulating the expression of NPAS2. In addition to contrast sensitivity, I also investigated the roles of NPAS2 and CLOCK in the regulation of electroretinogram (ERG) responses. In Clock-/- mice, the light-adapted ERG rhythm and dark-adapted ERG responses are significantly disrupted. In contrast, both light-adapted and dark-adapted ERG responses in Npas2-/- mice are not disrupted, suggesting that the functional role of NPAS2 outside of the GCL is limited.

Table of Contents

Chapter 1: Introduction 1

1.1 What are circadian rhythms? 2

1.2 Molecular mechanism of circadian rhythms 3

1.3 Circadian rhythms in the retina 7

1.3.1 The structure of the retina 8

1.3.2 Circadian rhythms in the retina 13

1.3.3 An autonomous clock in the retina 13

1.3.4 Localization of a clock in the retina 15

1.3.5 The implication of the dopamine pathway in the retinal clock mechanism 17

1.4 Project Objectives 22

1.5 References 25

Chapter 2: Description, justification, and validation of essential experimental procedures 39

2.1 A comparison of two methods of euthanasia on retinal dopamine levels 40

2.1.1 Descriptive Abstract 40

2.1.2 Introduction 41

2.1.3 Methods 42

2.1.4 Results 44

2.1.5 Discussion 45

2.2 Validation of the Specificity of Laser Capture Microdissection 48

2.2.1 Introduction 48

2.2.2 Methods 48

2.2.3 Results 49

2.3 Contrast sensitivity and optokinetic tracking (OKT) test 52

2.3.1 Contrast sensitivity 52

2.3.2 Optomotor Response/Optokinetic Tracking 56

2.4 The electroretinogram and circadian rhythm 58

2.4.1 What is an ERG? 58

2.4.2 Analyzing ERG responses 62

2.4.3 Studying cone or rod-specific ERG responses 62

2.4.4 Cone ERG responses are circadian 63

2.5 References 67

Chapter 3: Circadian rhythm of contrast sensitivity is regulated by a dopamine-NPAS2-adenylyl cyclase 1 signaling pathway in retinal ganglion cells 67

3.1 Abstract 73

3.2 Introduction 74

3.3 Materials and Methods 76

3.3.1 Animals 76

3.3.2 LacZ Histochemistry 76

3.3.3 Immunofluorescence imaging 77

3.3.4 Laser capture microdissection (LCM) 78

3.3.5 Quantitative real-time polymerase chain reaction (qRT-PCR) 79

3.3.6 Contrast sensitivity and visual acuity 79

3.3.7 Luciferase Reporter Assays and Transfections 80

3.3.8 Statistical analysis 82

3.4 Results 83

3.4.1 Contrast sensitivity exhibits circadian rhythmicity 83

3.4.2 Npas2 shows circadian expression in retinal ganglion cells 83

3.4.3 Contrast sensitivity is similarly reduced in Drd4-/-, Npas2-/-, and Adcy1-/- mice 85

3.4.4 Differential regulation of Adcy1 expression in Npas2-/- and Drd4-/- mice 87

3.4.5 Activation of the Adcy1 promoter by NPAS2/BMAL1 in NG108-15 cells 87

3.5 Discussion 89

3.6 Figures and Figure Legends 96

3.7 References 107

Chapter 4: Differential roles of retinal CLOCK and NPAS2 in mediating different dimensions of vision 117

4.1 Abstract 118

4.2 Introduction 119

4.3 Methods 121

4.3.1 Animals 121

4.3.2 Light Microscopy of Retinas 122

4.3.3 Laser capture microdissection (LCM) 122

4.3.4 Quantitative real-time polymerase chain reaction (qRT-PCR) 123

4.3.5 Luciferase Reporter Assays and Transfections 123

4.3.6 Contrast sensitivity and visual acuity 124

4.3.7 Retinal Function Test with Electroretinogram (ERG) 125

4.3.8 Statistical analysis 126

4.4 Results 127

4.4.1 Retinal morphology and structure preserved in young and old Clock-/- and Npas2-/- mice 127

4.4.2 CLOCK regulates the expression of Adcy1 and Npas2 transcripts in the ganglion cell layer 127

4.4.3 Selective Activation of the Adcy1 promoter by NPAS2/BMAL1 but not CLOCK/BMAL1 in NG108-15 cells 129

4.4.4 CLOCK is required for the circadian rhythm of contrast sensitivity 130

4.4.5 CLOCK regulates the circadian control of light-adapted ERG responses 130

4.4.6 CLOCK modulates dark-adapted ERG responses 131

4.5 Discussion 132

4.6 Figures and Figure Legends 138

4.7 References 146

Chapter 5: Summary and Future Directions 152

5.1 Summary 153

5.1.1 Retinal clocks regulate contrast sensitivity 153

5.1.2 Retinal clocks modulate ERG responses 157

5.2 Future Directions 158

5.2.1 The role of D4Rs and NPAS2 in the transcript rhythms of Drd4 and clock genes in the PRL 158

5.2.2 The role of NPAS2 in the retinal dopamine level and metabolism 160

5.2.3 The role of a dysfunctional clock in retinal diseases 162

5.2.4 Visual defects in night-shift workers 163

5.3 References 165

Appendix 167

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