Deposition of aggregated amyloid -protein (A), a proteolytic cleavage product of the amyloid precursor protein (1), is a critical step in the development of Alzheimer's disease (2). However, we are far from understanding the molecular mechanisms underlying the initiation of A polymerization in vivo. Here, we report that a seeding A, which catalyzes the fibrillogenesis of soluble A, is generated from the apically missorted amyloid precursor protein in cultured epithelial cells. Furthermore, the generation of this A depends exclusively on the presence of cholesterol in the cells. Taken together with mass spectrometric analysis of this novel A and our recent study (3), it is suggested that a conformationally altered form of A, which acts as a "seed" for amyloid fibril formation, is generated in intracellular cholesterol-rich microdomains.
A1 is physiologically secreted into the extracellular space; however, why and how soluble A aggregates and forms amyloid fibrils remains to be elucidated. A great deal of effort has been made to clarify this issue, using mainly in vitro systems. In most such experiments, it has been found that A at much higher concentrations than those prevailing in biological fluids is needed for A aggregation. Thus, it has been hypothesized that aggregation of soluble A involves seeded polymerization (4, 5), although this assumption has not yet been proved in vivo.We have recently reported the detection of a novel A in the apical compartment of cultures of MDCK cells that had been stably transfected with APP cDNA (⌬C MDCK cell) with a truncated cytoplasmic domain (⌬C APP) (3). This A species possesses unique molecular characteristics including its appearance as a smear on immunoblots and altered immunoreactivity. Significantly, these molecular characteristics disappeared dramatically following treatment of the cells with compactin or filipin, an inhibitor of de novo cholesterol synthesis and a cholesterol-binding drug, respectively. Based on previously reported evidence for ⌬C APP being missorted to the apical surface (6) and the cholesterol concentrations of the apical plasma membrane and apical transport vesicles being higher than those in other cellular membranes (7), we concluded that a novel A is generated from apically missorted APP in a cholesterol-dependent manner.Regarding the involvement of cellular cholesterol in the generation of the pathogenic protein, it must be noted that cholesterol-rich lipid microdomains within cells, called caveolae-like domains, have been reported to be the likely sites of the conversion of the normal cellular form of prion protein (PrP c ) to its pathogenic form (PrP sc ) (8 -11). Thus, it would be of great interest to investigate whether the novel A detected in our recent study (3), the generation of which is exclusively dependent on the presence of cholesterol in the cell, has the potential to act as a seed for fibrillogenesis of soluble A.
EXPERIMENTAL PROCEDURESMDCK Cell Culture-Dulbecco's modified Eagle's medium (Life Technologies, Inc...