Title Page
ABSTRACT
국문 초록
Contents
NOMENCLATURE 16
INTRODUCTION 19
CHAPTER 1. Study on the function of 14-3-3r in the cortical neurodevelopment 27
1.1. Chapter abstract 27
1.2. Chapter introduction 29
1.3. Materials and Methods 32
1.3.1. Animal 32
1.3.2. Genotyping 32
1.3.3. Real-time quantitative reverse transcription PCR 33
1.3.4. DNA construct 35
1.3.5. Immunoblotting 35
1.3.6. Immunohistochemistry 36
1.3.7. Primary culture 38
1.3.8. Immunocytochemistry 38
1.3.9. Stereotaxic microinjection for sparse labeling in vivo 39
1.3.10. Sholl analysis and neuronal morphological analysis 40
1.3.11. Genetic homogeneity analysis 40
1.3.12. Statistical analysis 41
1.4. Results 42
1.4.1. 14-3-3γ is highly conserved across the species in mammals. 42
1.4.2. 14-3-3γ haploinsufficiency causes developmental abnormalities. 42
1.4.3. 14-3-3γ haploinsufficient mice have reduced expression of 14-3-3γ. 47
1.4.4. 14-3-3γ affects cortical development. 50
1.4.5. 14-3-3γ haploinsufficiency results in abnormal cortical layer formation. 53
1.4.6. 14-3-3γ deficiency leads to abnormalities in the morphogenesis of cortical neurons in vitro. 56
1.4.7. 14-3-3γ deficiency leads to synaptic defects in excitatory cortical neurons in vitro. 61
1.4.8. 14-3-3γ haploinsufficiency results in morphological defects in cortical neurons in vivo. 64
1.4.9. 14-3-3γ haploinsufficiency alters the distribution of synaptic proteins in the cerebral cortex in vivo. 65
1.5. Discussion 69
1.6. 국문 초록 71
CHAPTER 2. Study on the function of 14-3-3r in the medial habenula 73
2.1. Chapter abstract 73
2.2. Chapter introduction 75
2.3. Materials and Methods 78
2.3.1. Animals 78
2.3.2. Quantitative real-time PCR 78
2.3.3. Co-immunoprecipitation 79
2.3.4. Immunoblotting 80
2.3.5. Immunohistochemistry 81
2.3.6. Bimolecular Fluorescence Complementation Experiment 82
2.3.7. DNA constructs 83
2.3.8. RNA sequencing 83
2.3.9. Sequencing data analysis 84
2.3.10. Bioinformatic analysis 85
2.3.11. Statistical analysis 86
2.4. Results 87
2.4.1. Changes in the mRNA expression of habenula-specific genes in the medial habenula of 14-3-3γ haploinsufficient mice 87
2.4.2. 14-3-3γ regulates the expression of OPRM1, a medial habenula-enriched G-protein receptor. 93
2.4.3. TCF7L2 is a putative transcription factor regulating OPRM1 expression. 96
2.4.4. TCF7L2 is a novel binding protein of 14-3-3γ. 99
2.4.5. TCF7L2 binds 14-3-3γ in a phosphorylation-dependent manner at specific phosphorylation sites. 99
2.4.6. 14-3-3γ regulates the translocation of TCF7L2 to the nucleus. 103
2.4.7. OPRM1 gene has TCF7L2 binding motifs near the exon1 promoter and in front of the exon11 promoter. 106
2.4.8. TCF7L2 regulates the expression of OPRM1. 109
2.5. Discussion 112
2.6. 국문 초록 116
CHAPTER 3. Study on the function of 14-3-3r in the dopaminergic pathway in the striatal and the substantia nigra 119
3.1. Chapter abstract 119
3.2. Chapter introduction 121
3.3. Materials and Methods 124
3.3.1. Animals 124
3.3.2. Genotyping 124
3.3.3. Quantitative reverse transcription-polymerase chain reaction 125
3.3.4. Western blotting 126
3.3.5. Immunohistochemistry 127
3.3.6. Quantitative dopamine measurement assay 128
3.3.7. Behavioral tests 129
3.3.8. Statistical analysis 133
3.4. Results 134
3.4.1. 14-3-3γ is mainly expressed in neurons in the striatum and substantia nigra of the brain. 134
3.4.2. Reduced 14-3-3γ levels affect dopamine metabolism. 139
3.4.3. 14-3-3γ reciprocally affects the phosphorylation of leucine-rich repeat kinase 2. 146
3.4.4. 14-3-3γ heterozygous knockout mice are implicated in astrogliosis. 150
3.4.5. 14-3-3γ heterozygous knockout mice display defects in motor coordination. 153
3.4.6. 14-3-3γ heterozygous knockout mice display defects in nest-building activity. 157
3.5. Discussion 160
3.6. 국문 초록 165
CONCLUSION 166
REFERENCES 174
CHAPTER 1 13
Figure 1-1. Construction and characterization of 14-3-3γ haploinsufficient mice. 44
Figure 1-2. Validation of 14-3-3γ expression level in 14-3-3γ haploinsufficient mice. 49
Figure 1-3. 14-3-3γ haploinsufficiency affects cortical layer formation. 55
Figure 1-4. 14-3-3γ deficiency causes impaired complexity of cortical neurons in vitro. 59
Figure 1-5. 14-3-3γ deficiency causes synaptic defects in cortical neurons in vitro. 63
Figure 1-6. 14-3-3γ haploinsufficiency leads to abnormalities in the morphogenesis of cortical neurons, resulting in synaptic defects in vivo. 67
CHAPTER 2 14
Figure 2-1. mRNA sequencing results of the medial habenula of 14-3-3γ heterozygous knockout mice and littermate wild-type mice. 90
Figure 2-2. Reduced expression of OPRM1 observed in the medial habenular of 14-3-3γ heterozygous knockout mice. 95
Figure 2-3. TCF7L2 is a habenular-specific transcription factor most closely related to genes with reduced expression in the medial habenular of mice and binds to... 98
Figure 2-4. 14-3-3γ binds to S156 of TCF7L2 in a phosphorylation-dependent manner. 102
Figure 2-5. 14-3-3γ promotes translocation of the transcription factor TCF7L2 to the nucleus. 105
Figure 2-6. The binding motif of TCF7L2 was found in the promoters of the OPRM1 gene. 108
Figure 2-7. TCF7L2 regulates the expression of endogenous OPRM1 in neuronal cell lines. 111
Figure 2-8. A model diagram that summarizes the overall results. 113
CHAPTER 3 15
Figure 3-1. YWHAG knockout heterozygous knockout mice were identified with reduced expression of 14-3-3γ. 138
Figure 3-2. Decreased expression of the phosphorylated tyrosine hydroxylase and insignificant change in expression of the tyrosine hydroxylase in the 14-3-3γ... 142
Figure 3-3. Decreased dopamine and dopamine transporter expression in the 14-3-3γ heterozygous knockout mice brain. 145
Figure 3-4. Decreased expression of the phosphorylated leucine-rich repeat kinase 2, but not total leucine-rich repeat kinase 2 expression in the 14-3-3γ heterozygous... 149
Figure 3-5. Increased expression of glial fibrillary acidic protein in the substantia nigra of the 14-3-3γ heterozygous knockout mice. 152
Figure 3-6. Defect in the motor coordination of 14-3-3γ heterozygous knockout mice without decreased basal motor activity. 156
Figure 3-7. Nest building impairment without a social deficit in the 14-3-3γ heterozygous knockout mice. 158
Supplementary Figure 1-1. The phenotype of 14-3-3γ haploinsufficient mice. 46
Supplementary Figure 1-2. 14-3-3γ is most expressed in the early postnatal period and affects cerebral cortex development. 51
Supplementary Figure 1-3. The 14-3-3γ is the most dominant isotype in primary cortical neurons, expressed at neuronal dendrites and synapses. 58
Supplementary Figure 2-1. (A) The model diagram for the functional characteristics of 14-3-3 protein, (B) the representative habenular neuronal circuit. 77
Supplementary Figure 2-2. Go analysis of mRNA sequencing results of the medial habenula of 14-3-3γ heterozygous knockout mice and littermate wild-type mice. 91
Supplementary Figure 3-1. Generation strategy and validation of YWHAG knockout mice. 136
Supplementary Figure 3-2. Measurement and quantification of immunofluorescence staining in the striatum and substantia nigra. 143