Intracellular Accumulation of β-Amyloid in Cells Expressing the Swedish Mutant Amyloid Precursor Protein

Abstract

␤-Amyloid (␤A) is a normal metabolic product of the amyloid precursor protein (APP) that accumulates in senile plaques in Alzheimer's disease. Cells that express the Swedish mutant APP (Sw-APP) associated with early onset Alzheimer's disease overproduce ␤A. In this report, we show that expression of Sw-APP gives rise to cell-associated ␤A, which is not detected in cells that express wild-type APP. Cell-associated ␤A is rapidly generated, is trypsin-resistant, and is not derived from ␤A uptake, indicating that it is generated from intracellular processing of Sw-APP. Intracellular and secreted ␤A are produced with different kinetics. The generation of intracellular ␤A is partially resistant to monensin and a 20°C temperature block but is completely inhibited by brefeldin A, suggesting that it occurs in the Golgi complex. Monensin, brefeldin A, and a 20°C temperature block almost completely inhibit ␤A secretion without causing increased cellular retention of ␤A, suggesting that secreted ␤A is generated in a post-Golgi compartment. These results suggest that the metabolism of Sw-APP gives rise to intracellular and secreted forms of ␤A through distinct processing pathways. Pathological conditions may therefore alter both the level and sites of accumulation of ␤A. It remains to be determined whether the intracellular form of ␤A plays a role in the formation of amyloid plaques.The deposition of ␤-amyloid (␤A) 1 in senile plaques is a pathological hallmark of Alzheimer's disease (Glenner and Wong, 1984;Masters et al., 1985). ␤A is a normal product of the metabolism of amyloid precursor protein (APP) in the secretory pathway Seubert et al., 1992;Shoji et al., 1992;Busciglio et al., 1993). Mutations in APP that are linked to autosomal dominant inheritance of Alzheimer's disease have been found to alter the production of ␤A. Mutation of the two amino acids proximal to the N terminus of ␤A has been described in individuals from two Swedish families that develop early onset Alzheimer's disease (Mullan et al., 1992). Expression of APP containing the Swedish mutation (Sw-APP) in transfected cells increases the production of ␤A by about 5-fold, suggesting a causal link between altered APP processing and the development of Alzheimer's disease (Citron et al., 1992;Cai et al., 1993). In this report, we show that the Swedish APP mutation not only increases ␤A production but also results in abnormal intracellular accumulation of ␤A. The processing pathways that give rise to intracellular and secreted ␤A can be distinguished by their differential kinetics and sensitivities to metabolic inhibitors and temperature block. EXPERIMENTAL PROCEDURESMetabolic Labeling and Immunoprecipitation-COS-1 cells maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) were transiently transfected with expressor plasmids encoding APP 695 , APP 751 , and APP 751 containing the Swedish mutation (KM changed to NL immediately N-terminal to the ␤A sequence, designated Sw-APP) (Mullan et al., 1992). HEK 293 cells ...

γ-Secretase Cleavage Site Specificity Differs for Intracellular and Secretory Amyloid β

“…These results are consistent with previously published data that A␤i generation was inhibited after BFA treatment in the mouse neuroblastoma cell line N2a and COS1 cells as well as in different cell lines transiently transfected with truncated APP constructs bearing an ER/IC retrieval signal, including kidney 293 cells and N2a cells (26,30,32,34). However, A␤42 generation was also found within the ER using very similar approaches (27,29,31).…”

“…Moreover, we obtained several lines of evidence that late Golgi compartments are involved in ␥-secretase processing. First, A␤i levels were dramatically increased when we blocked protein transport late in the secretory pathway using monensin, consistent with studies proposing the involvement of late Golgi compartments in ␥-secretase processing (26,32,34,84). Second, A␤i generation was increased in cells expressing a C99 mutant directed to the TGN.…”

“…Moreover, transgenic mice show accelerated neurodegeneration without extracellular amyloid deposition (24), indicating that intracellular amyloid-␤ peptides may play a crucial role in the development of AD. Intracellular sites with ␥-secretase activity were identified in primary neurons, neuronal cell lines, and peripheral cells (25)(26)(27)(28)(29)(30). Evidence has been obtained for the generation of A␤42 within the ER (27)(28)(29)31), where PS is predominantly localized (14 -16).…”

“…Evidence has been obtained for the generation of A␤42 within the ER (27)(28)(29)31), where PS is predominantly localized (14 -16). However, it has also been reported that A␤42 is produced in different organelles later in the secretory pathway and that the ER is not the major intracellular site of A␤42 generation (26,30,(32)(33)(34)74). Additionally, recent work has suggested that the A␤42 generation in the ER may be independent of PS (35).…”

Heat Shock Protein 70 Participates in the Neuroprotective Response to Intracellularly Expressed β-Amyloid in Neurons

“…Although no direct evidence has been provided, it has long been speculated that intraneuronal A␤ accumulation causes neuronal dysfunction, synaptic and neuronal loss, and dementia in AD and related Down's syndrome (LaFerla et al, 1995;Yang et al, 1998;Gouras et al, 2000;D'Andrea et al, 2001;Gyure et al, 2001;Busciglio et al, 2002). Several reports have documented intracellular accumulation of A␤ (Martin et al, 1995;Skovronsky et al, 1998;Gouras et al, 2000;Mochizuki et al, 2000;Takahashi et al, 2002b). A␤42 peptide appears to be the prominent form that accumulates inside neurons in AD brains and in transgenic mice expressing familial AD mutations (Takahashi et al, 2002a).…”