Title Page
ABSTRACT
Contents
ABBREVIATIONS 21
BACKGROUND 23
1. Alzheimer's disease 24
A. Prevalence and risk factors 24
B. Mechanism of Aβ and p-Tau production 25
C. Current treatment 25
D. Diabetes as a risk factor for AD 28
2. Endolysosomal network 32
A. Definition 32
B. ELN dysfunction as a contributing factor in the early stages of AD 32
C. The pathophysiological mechanism of ELN dysfunction in Aβ accumulation 33
D. Potential factors of ELN dysfunction 36
CHAPTER I. The effect of endosomal abnormalities induced by high glucose-mediated PICALM and mTORC1 on the modulation of amyloid precursor protein processing 46
INTRODUCTION 47
MATERIALS & METHODS 50
RESULTS 67
DISCUSSION 115
CHAPTER II. The effect of high glucose-mediated VPS26a downregulation on dysregulation of neuronal amyloid precursor protein processing and tau phosphorylation 122
INTRODUCTION 123
MATERIALS & METHODS 125
RESULTS 138
DISCUSSION 172
CHAPTER III. The protective effect of TRIM16-mediated lysophagy on high glucose-accumulated neuronal Aβ 180
INTRODUCTION 181
MATERIALS & METHODS 183
RESULTS 194
DISCUSSION 250
GENERAL CONCLUSION 257
REFERENCES 261
국문초록 293
Table 1. Body weight and blood glucose in ZLC and ZDF rats. 53
Table 2. Body weight and blood glucose in experimental mice. 54
Table 3. Human gene primers for real-time qPCR. 61
Table 4. The primer sequences used for real-time qPCR. 132
Table 5. The primer sequences used for methylation specific primer. 134
Table 6. List of primers used for real-time qPCR. 186
Figure 1. The pathophysiology of AD. 27
Figure 2. The potential mechanisms of DM-associated AD. 31
Figure 3. The potential mechanism of ELN dysfunction in Aβ accumulation. 35
Figure 4. The role of PICALM in clathrin-mediated endocytosis. 37
Figure 5. Structure and function of mTOR. 39
Figure 6. Retromer-mediated endosomal transport and its structure. 42
Figure 7. Molecular mechanism of lysophagy. 45
Figure 8. Early endosomal enlargement and amyloidogenesis are induced in the hippocampus of type 2 diabetic rodents. 69
Figure 9. Early endosomal enlargement and amyloidogenesis are induced in the hippocampus of type 1 diabetic rodents. 70
Figure 10. High glucose up-regulates Aβ and activates Rab5. 71
Figure 11. High glucose up-regulates Aβ through early endosomal enlargement. 72
Figure 12. High glucose facilitates APP endocytosis. 75
Figure 13. Lipid-raft mediated APP endocytosis induces early endosomal enlargement under high glucose conditions. 77
Figure 14. High glucose-induced ROS facilitates Sp1 nuclear translocation. 80
Figure 15. High glucose-stimulated Sp1 up-regulates the expression of endocytic proteins, and the levels of these proteins are increased in... 81
Figure 16. PICALM mediates recruitment of AP2A1 and CHC to APP and co-localizes with internalized APP. 85
Figure 17. PICALM mediates APP endocytosis, which up-regulates Aβ production. 87
Figure 18. Clathrin-dependent APP endocytosis induces early endosomal enlargement, which up-regulates Aβ production. 89
Figure 19. Autophagy is impaired in the hippocampus of diabetic rodents and SK-N-MCs exposed to high glucose. 93
Figure 20. PICALM induces autophagy under high glucose conditions. 95
Figure 21. High glucose-mediated AMPK/mTORC1 signaling impairs autophagy. 97
Figure 22. Defects in mTORC1-mediated autophagy impairs early endosomal clearance, which up-regulates Aβ. 99
Figure 23. Lysosomal dysfunction leads to endosomal clearance failure under high glucose conditions. 103
Figure 24. Protein levels of LAMP1 increases in the hippocampus of diabetic rodents and SK-N-MCs exposed to high glucose. 104
Figure 25. High glucose induced lysosomal dysfunction through ROS and mTORC1-mediated signaling. 106
Figure 26. High glucose-mediated cytosolic release of CTSB leads to Aβ accumulation. 107
Figure 27. Scheme of an in vivo experimental protocol using Dyansore or Rapamycin injection. 110
Figure 28. Increased early endosomes, Aβ increment, and cognitive impairment are recovered by pharmacological inhibition of clathrin-... 111
Figure 29. Increased early endosomes, Aβ increment, and cognitive impairment are recovered by pharmacological enhancement of... 114
Figure 30. Diagram of the pathways underlying the up-regulation of Aβ production by abnormalities in early endosomes, under high glucose conditions. 121
Figure 31. Body weight and blood glucose of experimental animals, and scheme of an in vivo experimental protocol using R33 treatment. 127
Figure 32. VPS26a is down-regulated in the hippocampus of STZ-diabetic mice. 140
Figure 33. VPS26a is down-regulated in neuronal cell exposed to high glucose. 142
Figure 34. High glucose upregulates DNMT1 through ROS/NF-κB signaling pathway. 146
Figure 35. DNMT1-mediated promoter hypermethylation down-regulates VPS26a under high glucose conditions. 148
Figure 36. R33 blocks elevated Aβ and p-Tau levels by high glucose. 150
Figure 37. R33 reverses the retention of APP to the early endosomes by high glucose in iPSC-NDs. 153
Figure 38. VSP26A overexpression reverses the retention of APP to the early endosomes by high glucose, which reduces Aβ. 154
Figure 39. High glucose has no significant alteration on the expression levels of tau phosphorylation-related proteins. 158
Figure 40. R33 reverses the retention of CI-MPR to the early endosomes by high glucose in iPSC-NDs, which increases CTSD... 160
Figure 41. VPS26A overexpression reverses the retention of CI-MPR to the early endosomes by high glucose, which increases CTSD... 162
Figure 42. VPS26A overexpression recovers high glucose-inhibited CTSD activity, which induces p-Tau degradation. 163
Figure 43. R33 reverses early endosomal enlargement, amyloidogenesis, and tau hyper-phosphorylation in diabetic mice. 167
Figure 44. R33 reverses synaptic deficits and astrocyte over-activation in diabetic mice. 170
Figure 45. R33 ameliorates cognitive impairment in diabetic mice. 171
Figure 46. The schematic model for dysregulation of mechanisms of APP processing and tau hyperphosphorylation by high glucose-... 179
Figure 47. High glucose reduces intact lysosomes in iPSC-NDs. 195
Figure 48. High glucose induces cytosolic release of CTSB and CTSD in both iPSC-NDs and hippocampal neurons. 196
Figure 49. High glucose induces Aβ accumulation in lysosomes and neuronal apoptosis in iPSC-NDs. 198
Figure 50. High glucose induces ROS -dependent lysosomal membrane permeabilization. 202
Figure 51. Recruitment of LC3, ubiquitin, and P62 to galectin-3 is blocked by high glucose. 203
Figure 52. Removal of galectin-3 puncta after lysosomal injury is inhibited by high glucose. 204
Figure 53. High glucose suppresses lysophagy. 205
Figure 54. Recovery of intact lysosomes after lysosomal injury is depend on lysophagy. 207
Figure 55. TRIM16 is down-regulated in iPSC-NDs, hippocampal neurons, and SH-SY5Ys exposed to high glucose. 211
Figure 56. TRIM16 is down-regulated in the hippocampus of diabetic mice. 213
Figure 57. High glucose elevates intracellular calcium levels and activates mTORC1. 214
Figure 58. Nuclear translocation of TFEB, but not TFE3, is inhibited by high glucose-mediated mTORC1 activation, and calcium influx has no... 216
Figure 59. High glucose suppresses the binding of TFEB to TRIM16 promoter. 218
Figure 60. Pharmacological inhibition of mTORC1 and enhancement of TFEB recover high glucose-inhibited TRIM16 levels. 220
Figure 61. Recruitment of TRIM16, not FBXO27, LRSAM1, and VCP, to the lysosome is inhibited under high glucose conditions. 224
Figure 62. Silencing of FBXO27, not LRSAM1 and VCP, inhibits lysophagy. 226
Figure 63. ALIX and CHMP4B recruitment to the lysosome is increased by high glucose. 228
Figure 64. TRIM16 overexpression reverses the decreased colocalization between LC3 and galectin-3 induced by high glucose. 229
Figure 65. TRIM16 overexpression reverses the decreased colocalization between p62 or ubiquitin and galectin-3 induced by high glucose. 231
Figure 66. TRIM16 overexpression induces removal of galectin-3 signal intensities inhibited by high glucose after lysosomal injury. 233
Figure 67. TRIM16 overexpression recovers lysotracker signal intensities inhibited by high glucose. 234
Figure 68. TRIM16 overexpression reverses the increased colocalization between Rab7 and LAMP1 induced by high glucose. 236
Figure 69. TRIM16 overexpression recovers activities of lysosomal hydrolases and autophagy inhibited by high glucose. 238
Figure 70. TRIM16 overexpression degrades high glucose-accumulated Aβ and p-Tau, and prevents cell death. 239
Figure 71. Scheme of an in vivo experimental protocol using curcumin C1 treatment, and body weight and blood glucose of experimental animals. 242
Figure 72. Curcumin C1 reverses TFEB nuclear translocation inhibited in the hippocampus of diabetic mice. 243
Figure 73. Curcumin C1 recovers lysophagy inhibited in the hippocampus of diabetic mice. 244
Figure 74. Curcumin C1 ameliorates cytosolic release of lysosomal hydrolases induced in the hippocampus of diabetic mice. 245
Figure 75. Curcumin C1 inhibits Aβ and p-Tau accumulation in the hippocampus of diabetic mice. 247
Figure 76. Curcumin C1 improves cognitive impairment induced in diabetic mice. 248
Figure 77. The schematic model for effects and molecular mechanism of action of high glucose on neuronal lysopahgy and subsequent... 256
Figure 78. The schematic model summarizing the proposed pathway in high glucose-induced neuronal Aβ accumulation through... 260