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)

Permanent URL: https://etd.library.emory.edu/concern/etds/gf06g302s?locale=en
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Abstract

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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