Caveolin-1 調節γ-secretase 的分子機制之研究

Abstract

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.