Title Page
Contents
CHAPTER Ⅰ. INTRODUCTION 14
1. Blood-Brain Barrier 14
2. Alzheimer's Disease 16
3. Receptor-mediated Transcytosis 22
4. Dab-1 like PTB adaptor protein 24
5. My Research Purpose 27
CHAPTER Ⅱ. ANKS1A regulates the LDL receptor related protein 1 (LRP1)-mediated cerebrovascular clearance pathway 29
1. ABSTRACT 29
2. INTRODUCTION 30
3. MATERIALS AND METHODS 34
3.1. Mice 34
3.2. Mouse brain primary cell culture 35
3.3. In vitro BBB studies 36
3.4. X-gal staining and immunostaining analysis 38
3.5. Western blot 40
3.6. RNA analysis 40
3.7. Modified Y-maze test (spatial reference memory) 41
3.8. Vascular β-amyloid ELISA 41
3.9. In vivo administration and viral transduction with AAV vectors 43
3.10. Image analysis 43
3.11. Statistics 46
4. Results 52
4.1. Regulation of the brain endothelial surface LRP1 by ANKS1A in the bEnd.3 cellular barrier model of BBB 52
4.2. Regulation of the brain endothelial LRP1 by ANKS1A in in vivo mouse models 75
4.3. Mechanism underlying the regulation of endothelial LRP1 by ANKS1A 85
4.4. Investigation of β-amyloid accumulation and vascular integrity in ANKS1A cKO; 5XFAD 91
4.5. Rescue of the defective vascular clearance by the brain endothelial ANKS1A gene delivery 97
5. Discussion 118
REFERENCES 124
ABSTRACT IN KOREAN 149
CHAPTER Ⅲ. INTRODUCTION 154
1. Cerebral Cavernous Malformation (CCM) 154
2. Endothelial-Mesenchymal Transition (Endo-MT) 156
CHAPTER Ⅳ. ANKS1A may play a role in maintaining or stabilizing healthy blood vessels in the brain during aging 158
1. ABSTRACT 158
2. Introduction 159
3. Methods 161
3.1. Mice 161
3.2. X-gal staining and immunostaining analysis 162
3.3. Image analysis 163
3.4. Evans blue extravasation assay 164
3.5. Primary mouse brain endothelial cell culture 164
3.6. Quantification 165
3.7. Statistics 165
4. RESULT 166
4.1. The specific expression of ANKS1A in the brain endothelial cells 166
4.2. CCM-like vessel lesions in the aged ANKS1A KO brain 169
4.3. Immune cells are focally increased around the CCM-like lesion 173
4.4. CCM-like lesions in the aged ANKS1A brain exhibit increased mesenchymal markers 177
5. Discussion 182
REFERENCES 184
ABSTRACT IN KOREAN 193
Table 1. Primer sequence used in Genotyping 47
Table 2. siRNA sequence used Knock-down assay 48
Table 3. Antibody used in Immunostaining 49
Table 4. Primer sequence used in qRT-PCR assay 50
CHAPTER Ⅰ. INTRODUCTION 9
Figure 1. Anatomical Structure of the Blood-Brain Barrier (BBB) 15
Figure 2. Pathways of Amyloid Precursor Protein (APP) processing and Alzheimer's Disease pathogenesis 18
Figure 3. The structure of Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1) 21
Figure 4. Pathways of β-Amyloid clearance across the brain capillary endothelium 23
Figure 5. The role of ANKS1A in Receptor Tyrosine Kinase (RTK) transport from the ER 26
CHAPTER Ⅱ. ANKS1A CHAPTER II. ANKS1A regulates the LDL receptor related protein 1 (LRP1)-mediated cerebrovascular clearance pathway 9
Figure 6. ANKS1A level is correlated to LRP1 level in BBB 54
Figure 7. ANKS1A is specifically expressed in cerebrovascular endothelial cells 56
Figure 8. ANKS1A is up-regulated in brain microvessels 60
Figure 9. Brain endothelial expression of ANKS1A is regulated by the integrity of BBB 62
Figure 10. Cell surface level of LRP1 is reduced in ANKS1A-deficientbEnd.3 cells 66
Figure 11. In vitro BBB setup using bEnd.3 cell 68
Figure 12. ANKS1A-deficient bEnd.3 cell shows that the cerebrovascular clearance is impaired 70
Figure 13. EC-specific ANKS1A loss does not disrupt the cerebrovascular integrity and BBB 74
Figure 14. Surface level of LRP1 is reduced in ANKS1A cKO brain 78
Figure 15. Surface level of LRP1 is reduced in ANKS1A cKO brain 80
Figure 16. β-amyloid clearance is reduced in the microvessels of ANKS1A cKO brain 82
Figure 17. β-amyloid is reduced in the microvessels of ANKS1A cKO brain 84
Figure 18. ANKS1A interacts with two NPXY motifs in the LRP1 receptor 88
Figure 19. ANKS1A promotes the anterograde transport of LRP1 in the ER 90
Figure 20. β-amyloid clearance is impaired in ANKS1A cKO brain 94
Figure 21. Cerebrovascular patterning was disturbed in ANKS1A cKO 96
Figure 22. Vascular β-amyloid plaques (CAA) are more increased in ANKS1A KO; 5XFAD 99
Figure 23. Vascular β-amyloid plaques (CAA) are more increased in ANKS1A KO; 5XFAD 101
Figure 24. ANKS1A loss induces a cerebrovascular breakdown through impaired β-amyloid clearance 103
Figure 25. ANKS1A loss induces a cerebrovascular breakdown through impaired β-amyloid clearance 105
Figure 26. Increased leakage of peripheral blood into parenchymal tissues in ANKS1A KO;5XFAD 107
Figure 27. In vivo administration of AAV-BR1 vector (Brain microvessel-specific AAV) 111
Figure 28. The AAV-BR1-ANKS1A virus injection rescued β-amyloid burden and behavioral impairment of ANKS1A KO mice 113
Figure 29. The AAV-BR1-ANKS1A virus injection rescued the number of neurons and neurites in ANKS1A KO mice 115
Figure 30. Final Model 117
CHAPTER Ⅲ. INTRODUCTION 11
Figure 31. Progression of cerebral cavernous malformation (CCM) lesion pathogenesis. 155
Figure 32. Progression of Endothelial to Mesenchymal Transition. 157
CHAPTER Ⅳ. ANKS1A may play a role in maintaining or stabilizing healthy blood vessels in the brain during aging 12
Figure 33. Brain vessel patterning is not impaired by ANKS1A deficiency in adult brains 168
Figure 34. Brain lesions that resemble CCM are present in aged ANKS1A KO mice 171
Figure 35. Aged ANKS1A KO brains exhibit increased immune cells 175
Figure 36. The Aged ANKS1A brains show an increased mesenchymal marker expression 179
Figure 37. Final model 181