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베타-시크리테아제 발현 조절 기전에 관한 연구

Studies on the Regulation Mechanism of Beta-Secretase Expression

초록/요약

Alzheimer’s disease (AD) is the most common form of senile dementia characterized by senile plaques, neurofibrillary tangles and neuronal loss. Aβ peptide, a major component of senile plaques, is involved in the pathological events that result in the clinical symptoms of AD. Prominent inflammatory response and disruption of intracellular calcium homeostasis are also observed in AD pathogenesis. BACE1 (β-site APP cleaving enzyme) is an essential enzyme for Aβ generation. It has been reported that the levels of BACE1 expression and enzymatic activity are elevated in sporadic AD cases compared to age-matched normal brains. In this study, we hypothesized that BACE1 expression is regulated at the transcriptional level by dissecting BACE1 promoter. We found IFN-γ, pro-inflammatory cytokine, activated JAK2 and ERK1/2 and then phosphorylated STAT1 binds to the putative STAT1 binding sequences in BACE1 promoter region, resulting in modulating BACE1 gene expression in astrocytes. In late-onset of AD, aging is the most important risk factor and contributes to increasing in abnormal accumulation of Aβ and the disruption of calcium homeostasis. We examined the role of NFAT1, which is a transcription factor regulated in neurons. Treatment of calcium ionophore or Aβ, which is known to elevate intracellular calcium level, elevated BACE1 expression via calcineurin/NFAT1 signaling pathway. Since expression of RAGE (receptor for advanced glycation endproducts), a multiligand receptor for AGEs and Aβ, is elevated in the brains of aged individuals as well as AD patients, the role of RAGE in BACE1 expression was examined. We found that NFAT1 activation and BACE1 expression were enhanced in RAGE overexpressing neurons. Taken together, BACE1 expression was regulated at the transcription level in the pathological environment of AD. It suggests possible targets to develop Aβ-lowering drugs for AD pathogenesis.

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목차

ABSTRCT i
LIST OF CONTENTS iii
LIST OF FIGURES vii
ABBREVIATION ix
I. INTRODUCTION 1
1. Amyloid precursor protein (APP) processing 2
A. Non-amyloidogenic processing 2
B. Amyloidogenic processing 4
2. Secretase 4
A. Alpha-secretase 4
B. Beta-secretase 6
C. Gamma-secretase 7
3. Neuroinflammation 9
A. Inflammation in AD 9
B. BACE1 and inflammation 11
C. STAT1 signaling pathway 11
4. Calcium homeostasis 15
A. Calcium disruption in AD 15
B. Calcineurin/NFAT signaling pathway 16
5. Aging in SAD 17
A. Level of BACE1 in SAD 19
B. RAGE 19

II. METERIALS AND METHODS 24
1. Cell line and Primary cortical culture 24
2. Reagent treatment 24
3. Cloning and cDNA constructs 25
4. Transfection and Luciferase assay 26
5. Preparation of cell extracts 26
6. Antibodies 27
7. Electrophoretic mobility shift assay (EMSA) 28
8. Nuclear extracts preparation for EMSA 29
9. RNA isolation and RT-PCR 29
10. ICV and stereotaxic injection into mouse brains 29
11. RAGE-adenovirus ICV injection into the Tg2576 mouse brain 30
12. Immunostaining 30
13. Constructs of RAGE adenovirus 31
14. Sandwich ELISA for Aβ quantification 31
15. Statistical analysis 31

III. RESULTS 32
1. IFN-γ-induced BACE1 expression via JAK/STAT signals in astrocytes 32
A. Injection of IFN-γ stimulated BACE1 protein expression in astrocytes 32
B. Injection of IFN-γ had no effects on neuronal BACE1 expression in vivo 37
C. The human BACE1 gene promoter region was identified
40
D. BACE1 promoter was responsible for IFN-γ stimulation 43
E. STAT1 directly bound to specific sequences of BACE1 promoter 46
F. STAT1 binding sequence deleted mutant showed no effects of IFN-γ 50
G. SOCS blocked IFN-γ-induced BACE1 promoter activity in astrocytes 52
H. IFN-γ-induced BACE1 expression was blocked by PD98059 in astrocytes 55
I. IFN-γ-activated ERK1/2 phosphorylated serine site of STAT1 58

2. BACE1 expression by calcineurin/NFAT1 signals in neuron 61
A. Ionomycin facilitated BACE1 gene expression 61
B. NFAT1 interacted with specific site in the BACE1 promoter 65
C. NFAT1, but not NFAT4, regulated BACE1 gene expression 68
D. Aβ1-42 treatment induced NFAT1 activation 71
E. Aβ1-42 up-regulated BACE1 expression via activation of NFAT1 75
F. Calcineurin/NFAT1 regulated BACE1 expression in primary cortical cells 78
G. NFAT1 protein were activated in Tg2576 mouse brains 81
3. Increased BACE1 level in RAGE expressing neuron via NFAT1 activation 84
A. NFAT1 activation was increased in aged mouse brains 84
B. AGE induced NFAT1 activation via RAGE expression 87
C. BACE1 expression was elevated by AGE treatment 90
D. Overexpression of RAGE activated NFAT1 92
E. Overexpression of RAGE elevated BACE1 expression 95
F. Aβ-induced BACE1 promoter and NFAT1 activation were elevated by RAGE 99
G. Aβ up-regulated BACE1 via RAGE expression 102
H. Soluble RAGE blocked Aβ-induced BACE1 expression in RAGE-SY5Y 104
I. RAGE enhanced BACE1 expression in mouse primary cortical cells 107
J. Aβ generation were regulated by RAGE injection via BACE1 expression 110

IV. DISCUSSION 115
1. IFN-γ-induced BACE1 expression via JAK/STAT signals in astrocytes 115
2. BACE1 expression by calcineurin/NFAT1 signals in neuron 119
3. Increased BACE1 level in RAGE expressing neuron via NFAT1 activation 122

REFERENCES 126

국문요약 153

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