Effects and Possible Mechanisms of Some Phytochemicals on Drosophila Models of Alzheimers Disease
식물유래 화합물의 알츠하이머 초파리 모델에 대한 효과 및 작용메카니즘

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dc.description학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2016. 2. 안용준.-
dc.description.abstractAlzheimer's disease (AD) is the most common type of presenile and senile dementia in developed and developing countries. The human β-amyloid (Aβ) cleaving enzyme (BACE-1) is a key enzyme responsible for amyloid plaque production, which implicates the progress and symptoms of AD. In this study, a fluorescence resonance energy transfer (FRET)-based enzyme assay was used to identify the BACE-1 inhibitory constituents from methanol extracts from the rhizomes of turmeric, Curcuma longa L. (Zingiberaceae), and the whole blue licorice (Korean mint) plant, Agastache rugosa (Fisch. & C.A. Mey.) O. Kuntze (Lamiaceae). The active constituents were determined to be the curcuminoids, diarylalkyls curcumin (CCN), demethoxycurcumin (DMCCN) and bisdemethoxycurcumin (BDMCCN) from C. longa rhizomes, and the O-methylated flavone, acacetin, and the oleanane triterpenoids, maslinic acid and oleanolic acid from whole A. rugosa plants. BDMCCN exhibited the strongest inhibitory activity toward BACE-1 with IC50 17 μM, which was 20 and 13-fold more potent than those of CCN and DMCCN, respectively. Quantitative structure–activity relationship of the curcuminoids indicates that structural characteristics, such as degrees of saturation, types of carbon skeleton and functional group, and hydrophobicity rather than molecular weight appear to play a role in determining inhibitory potency of curcuminoids on BACE-1. Acacetin was a 4.0-fold and 5.5-fold more potent inhibitor of BACE-1 than oleanolic acid and maslinic acid, respectively. Overall, these compounds were significantly less potent inhibitors of BACE-1 than a positive control, the cell-permeable isophthalamide, BACE-1 inhibitor IV.
To assess the neuro-protective ability of the curcuminoids and acacetin, the Drosophila melanogaster models of AD were constructed and characterized by phenotypes, histological analysis, and reverse transcription polymerase chain reaction (RT-PCR). Drosophila model system overexpressed BACE-1 and its substrate amyloid precursor protein (APP) in compound eyes and entire neurons. Overexpression of APP/BACE-1 resulted in the progressive and measurable defects in morphology of eyes and locomotion. The feeding, climbing activity, lifespan, and morphological changes in fly eyes were also evaluated. Remarkably, supplementing diet with either BDMCCN, CCN, and acacetin rescued APP/BACE-1-expressing flies and kept them from developing both eye morphology (dark deposits, ommatidial collapse and fusion, and the absence of ommatidial bristles) and behavioral (motor abnormalities) defects.
Acacetins mechanisms of action on transgenic Drosophila model of AD were also determined. The real-time RT-PCR analysis revealed that acacetin reduced both the human APP and BACE-1 mRNA levels in the transgenic flies, suggesting that it plays an important role in the transcriptional regulation of human BACE-1 and APP. Western blot analysis revealed that acacetin reduced Aβ production by interfering with BACE-1 activity and APP synthesis, resulting in a decrease in the levels of the APP carboxy-terminal fragments and the APP intracellular domain. Therefore, the protective effect of acacetin on Aβ production is mediated by transcriptional regulation of BACE-1 and APP, resulting in decreased APP protein expression and BACE-1 activity. Acacetin also inhibited APP synthesis, resulting in a decrease in the number of amyloid plaques.
In conclusion, C. longa rhizome-derived curcuminoids and whole A. rugosa plant- derived acacetin are potential therapeutics or lead compounds for the prevention or treatment of AD. The anti-AD action of these compounds provides an indication of at least one of the pharmacological actions of C. longa and A. rugosa. Detailed tests are needed to understand how to improve the anti-AD potency and stability of the compounds isolated from C. longa and A. rugosa for eventual commercial development.
dc.description.tableofcontentsINTRODUCTION 1

1. Alzheimers disease 4
1.1. Introduction of Alzheimers disease 4
1.2. Pathological hallmarks of Alzheimers disease 4
1.3. Epidemiology and worldwide death rate of Alzheimers disease 5
1.4. Risk factors and causes for Alzheimers disease 6
1.4.1. Age 6
1.4.2. Family history and genetic factors 6
1.4.3. Nongenetic factors 8
1.5. Market and cost of Alzheimers disease 8
1.6. Treatments for Alzheimers disease 9
1.7. Hypotheses involved in the pathological processing of Alzheimers disease 9
1.7.1. Cholinergic hypothesis of Alzheimers disease 9
1.7.2. Amyloid cascade hypothesis of Alzheimers disease 12
1.7.3. Tau hypothesis 13
2. Amyloid precursor protein and its proteolytic products 14
2.1. Basic knowledge of amyloid precursor protein 14
2.2. APP proteolytic processing 15
3. BACE-1 (β-Secretase 1) 17
3.1. Basic knowledge of BACE-1 17
3.2. BACE-1 structure and catalytic mechanism 18
3.3. In vitro BACE-1 FRET assay principle 19
3.4. BACE-1 inhibitors 20
4. Drosophila as a model for Alzheimers disease 22
4.1. Advantages of Drosophila as a model for human disease 22
4.2. Drosophila models of Alzheimers disease 25
4.2.1. Ortholog of human APP, α-secretase, and β-secretase, and the component ofγ-secretase in Drosophila 25
4.2.2. Gal4-UAS system and Drosophila models of Alzheimers disease 26
5. Curcuma longa 33
6. Agastache rugosa 33
7. Perspectives 34

CHAPTER I BACE-1 Inhibitory Phytochemicals Identified from Curcuma longa Rhizomes and Whole Agastache rugosa Plants 36
1.1. Materials and reagents 38
1.2. Plants 39
1.3. Instrumental analysis 39
1.4. FRET enzyme assay 40
1.5. Bioassay-guided fractions and isolation 40
1.5.1. Curcuma longa rhizomes 40
1.5.2. Whole Agastache rugosa plants 45
1.6. Data analysis 51
1.1. FRET bioassay-guided fractionation and isolation 52
1.1.1. Curcuma longa rhizomes 52
1.1.2. Whole Agastache rugosa plants 62
1.2. In vitro BACE-1 inhibitory activity of isolated compounds 76
1.2.1. BACE-1 inhibitory activity of compounds from Curcuma longa rhizomes 76
1.2.2. BACE-1 inhibitory activity of compounds from whole Agastache rugosa plants 77

CHAPTER II Effects of Phytochemicals on Behavior, Eye Morphology, and Lifespan of Drosophila Models of Alzheimers Disease 80
2.1. Materials and reagents 82
2.2. Drosophila stocks and rearing conditions 82
2.3. Experimental groups 82
2.3.1. Curcuminoids supplementation 82
2.3.2. Acacetin supplementation 83
2.4. Light and scanning electron microscopy of the adult eyes 86
2.5. Histologic analysis 86
2.6. RT-PCR analysis of human APP and BACE-1 genes in transgenic fly 87
2.7. Measurement of the fly eclosion rate 88
2.8. Lifespan assay 88
2.9. Climbing assay 89
2.10. Feeding assay 89
2.11. Data analysis 90
2.1. Characterization of trans-human APP and BACE-1 genes fly as reliable models of Alzheimers disease 91
2.2. Effects of curcuminoids on behavior, eye morphology and lifespan of Drosophila models of AD 93
2.2.1. Effects on the eye morphology 94
2.2.2. Effects on climbing behaviors 96
2.2.3. Effects on lifespan and feeding 99
2.3. Effects of acacetin on behavior, eye morphology and lifespan of Drosophila models of AD 104
2.3.1. Effect on age-dependent neurodegeneration, as reflected by an aberrant eye phenotype 104
2.3.2. Acacetin has a protective effect on age-dependent neurodegeneration of the eye 110
2.3.3. Effect on the flies age-dependent motor abnormalities 112
2.3.4. Effect on fly feeding and longevity 114
2.3.5. Effect on eclosion of the transgenic flies 116

CHAPTER III Possible Mechanisms of Action of Acacetin on Drosophila Model of Alzheimers Disease 122
3.1. Materials and reagents 124
3.2. Antibodies 124
3.3. Drosophila stocks and rearing conditions 124
3.4. Experimental groups 125
3.5. Real-time reverse transcription-PCR analysis 125
3.6. Western blot analysis 126
3.7. Data analysis 127
3.1. Effect of acacetin on the human APP and BACE-1 mRNA levels 128
3.2. Acacetin significantly reduces the Aβ levels by interfering with human APP proteolytic processing and BACE-1 expression 130


dc.format.extent7271391 bytes-
dc.publisher서울대학교 대학원-
dc.subjectAlzheimer’s disease-
dc.subjectDrosophila melanogaster-
dc.subjectCurcuma longa-
dc.subjectAgastache rugosa-
dc.subjectmechanisms of action-
dc.titleEffects and Possible Mechanisms of Some Phytochemicals on Drosophila Models of Alzheimers Disease-
dc.title.alternative식물유래 화합물의 알츠하이머 초파리 모델에 대한 효과 및 작용메카니즘-
dc.contributor.AlternativeAuthorXue Wang-
dc.contributor.affiliation농업생명과학대학 농생명공학부-
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Ph.D. / Sc.D._농생명공학부)
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