臺灣博碩士論文加值系統

越來越多證據指出，amyloid-precursor protein (APP)的β以及γ的水解可能是發生在細胞膜上一種稱為lipid rafts 的特殊構造中，而caveolin-1 是此種構造上一個重要的結構蛋白。此外，在阿茲海默氏症病患腦部的海馬迴區域，caveolin-1的表現有上升的現象。這些結果都顯示caveolin-1 有可能參與在調節APP 的分解以及阿茲海默氏症的致病過程中。因此，我們嘗試去證明caveolin-1 在γ-secretase主導的蛋白質水解上扮演關鍵的角色。在本實驗中，我們觀察到在HEK293 細胞裡，大量表現的caveolin-1 會促進raft membrane 的形成以及刺激γ-secretase 對於APP 的分解。然而，在γ-secretase 的另外一個作用對象Notch 身上，偵測不到此種情況。這些結果顯示caveolin-1 可以調控γ-secretase 對於受質的選擇性。接著，利用MβCD 這種抽除膽固醇的藥物去破壞raft membrane，可以完全抑制caveolin-1 所增加的γ-secretase 活性，由此更進一步證明了細胞膜形成特殊結構這個現象。另外，我們還發現APP，nicastrin，和ERK 會與caveolin-1 共同出現在raft membrane 裡。同時，caveolin-1 被報導可以抑制ERK 的活化，而我們確實也發現caveolin-1 藉此間接地促進γ-secretase 對於APP 的水解作用。這正好可以呼應我們之前的研究結果:降低ERK 的活化可以增加γ-secretase 的活性。接下來，我們抑制dynamin-2 的表現來阻斷細胞內噬作用，觀察到caveolin-1 對於γ-secretase 的刺激作用有顯著的降低。最後，我們發現大量表現的caveolin-1 也
會增加Aβ的產生，而抑制細胞內噬作用，同樣可以阻止caveolin-1 造成的影響。總結來說，透過以上的研究，我們相信caveolin-1 在調節γ-secretase 分解APP 的活性以及阿茲海默氏症的致病過程上確實扮演了重要的角色。

Accumulated evidence has suggested that β- and γ-cleavage of amyloid precursor protein (APP) may take place in specialized membrane microdomains called lipid
rafts. Caveolin-1 is an important structure protein of this membrane domain, and its expression in the hippocampus region of Alzheimer’s disease patients’ brains is upregulated. These data all imply that caveoiln-1 could play a role in modulating the proteolytic processing of APP and the pathogenesis of Alzheimer’s disease. We thus seek to determine whether caveolin-1 can actively involve in the regulation of γ-secretase-mediated proteolysis. In this study, we observe that overexpression of caveolin-1 promotes the formation of raft-membranes in HEK293 cells and concomitantly stimulates γ-secretase-mediated cleavage of APP. However, the γ-secretase-mediated S3 cleavage of Notch, another substrate of γ-secretase, is not affected by the overexpression of caveolin-1, suggesting that substrate selectivity of
γ-secretase can be modulated by caveolin-1. Consistently, the caveolin-1-enhanced γ-secretase activity can be completely abolished by a cholesterol-depleting drug called
MβCD that also destabilizes the raft membranes. This strengthen the notion that caveolin-1 can promote the formation of raft membrane. We further confirm that APP,
nicastrin, and ERK are co-localized with caveolin-1 in raft membranes. Caveolin-1 is also known to be a scaffold protein that can suppress ERK activation. In caveolin-1-overexpressed cells, the evidence that the suppression of ERK activation is prominent in accordance to our previous findings that down-regulation of ERK can promote γ-secretase activity. And then, the down-regulation of dynamin-2, a critical component of endocytosis, results in a significant decrease in caveolin-1-enhanced γ-secretase activity. Finally, we discover that overexpression of caveolin-1 also can increases Aβ production, and inhibition of endocytosis blocks this effect. Together,
the present study strongly suggests that caveolin-1 plays an important role in the regulation of γ-secretase-catalyzed proteolysis of APP and may exacerbate the
pathogenesis of Alzheimer’s disease.

Table of contents I
List of Figures III
摘要 1
Abstract 3
Introduction 5
Alzheimer’s disease 5
Aβ generation and γ-secretase 6
Lipid rafts and secretase-mediated proteolysis of APP 9
Caveolin and its relationship with AD 10
Objectives and hypothesis 12
Materials and methods 14
Reagents 14
Cell culture 15
Generation of DNA Constructs 15
Transient transfection of mammalian cells 16
Cell-based γ-secretase assays 16
SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis 18
Transfection of small interfering RNAs (siRNAs) targeting dynamin-2 19
Aβ ELISA 20
Membrane fractionation 20
Results 23
The expression of exogenous caveolin-1 facilitates the formation of raft-membranes 23
Overxepression of caveolin-1 increases γ-secretase-mediated cleavage of APP, while Notch processing seems unchanged. 24
Caveolin-1-induced formation of raft microdomains is a structurally critical for the enhancement of γ-secretase activity and the recruitment of this protease and APP 25
Endocytosis plays a crucial role in the caveolin-1-elicited stimulation of γ -secretase activity 27
Discussion 30
Differential processing between APP and Notch 30
Caveolin-1-mediated endocytosis and APP proteolysis 32
Aβ production 33
Summary 34
Reference 35
List of Figures
Figure 1. Tetracycline-inducible expression of caveolin-1 and the quantitative measurement of γ-secretase activity. 43
Figure 2. The tetracycline-inducible expression of caveolin-1 is functional in the formation of raft-membranes. 44
Figure 3. Overxepression of caveolin-1 increases γ-secretase-mediated cleavage of APP. 45
Figure 4. Overexpression of caveolin-1 dose not affect Notch processing. 47
Figure 5. Cholesterol depletion completely abolishes caveolin-1-induced γ-secretase activity. 48
Figure 6. Overexpression of caveolin-1 recruits APP, mature nicastrin, and ERK into raft-fraction. 49
Figure 7. Overexpression of caveolin-1 suppresses ERK activation. 50
Figure 8. Inhibition of endocytosis decreases caveolin-1-stimulated γ-secretase activity. 52
Figure 9. Overexprssion of caveolin-1 increases extracellular Aβ, which can be blocked by the inhibition of endocytosis. 53
Figure 10. Model for the molecular mechanisms depicting caveolin-1-elicited regulation of γ-secretase. 54

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