Down-regulation of Endogenous Amyloid Precursor Protein Processing due to Cellular Aging

Kai Prager,Christian Behl,Jochen Walter,Andreas Kern,Birgit Roempp

doi:10.1074/jbc.m505625200

Abstract

Processing of amyloid precursor protein (APP) is a well acknowledged central pathogenic mechanism in Alzheimer disease. However, influences of age-associated cellular alterations on the biochemistry of APP processing have not been studied in molecular detail so far. Here, we report that processing of endogenous APP is down-regulated during the aging of normal human fibroblasts (IMR-90). The generation of intracellular APP cleavage products C99, C83, and AICD gradually declines with increasing life span and is accompanied by a reduced secretion of soluble APP (sAPP) and sAPPalpha. Further, the maturation of APP was reduced in senescent cells, which has been shown to be directly mediated by age-associated increased cellular cholesterol levels. Of the APP processing secretases, protein levels of constituents of the gamma-secretase complex, presenilin-1 (PS1) and nicastrin, were progressively reduced during aging, resulting in a progressive decrease in gamma-secretase enzymatic activity. ADAM10 (a disintegrin and metalloprotease 10) and BACE (beta-site APP-cleaving enzyme) protein levels exhibited no age-associated regulation, but interestingly, BACE enzymatic activity was increased in aged cells. PS1 and BACE are located in detergent-resistant membranes (DRMs), well structured membrane microdomains exhibiting high levels of cholesterol, and caveolin-1. Although total levels of both structural components of DRMs were up-regulated in aged cells, their particular DRM association was decreased. This age-dependent membrane modification was associated with an altered distribution of PS1 and BACE between DRM and non-DRM fractions, very likely affecting their APP processing potential. In conclusion, we have found a significant modulation of endogenous APP processing and maturation in human fibroblasts caused by age-associated alterations in cellular biochemistry.

Highlights

At least three amyloid precursor protein (APP) processing secretases are identified
Processing of endogenous APP throughout the in vitro life span of the Normal human fibroblasts (NHFs) culture was analyzed by monitoring the protein levels of fulllength APP, the intracellular cleavage products C99, C83, and APP intracellular domain (AICD), as well as the secreted cleavage products soluble APP (sAPP) and sAPP␣ at increasing population doubling levels (PDLs) by immunoblotting (Fig. 1)
We observed a gradual down-regulation of endogenous APP processing during in vitro aging of NHFs

Summary

EXPERIMENTAL PROCEDURES

Cellular Aging Down-regulates APP Processing becco’s modified Eagle’s medium (Invitrogen) supplemented with antibiotics (Invitrogen), 1 mM sodium pyruvate (Invitrogen), and 10% charcoal-dextran-treated fetal calf serum (HyClone) These deficient culture conditions accelerate aging without influencing the resulting general aged phenotype, as described previously [22]. According to the cell lysate protein concentration, the medium was loaded on an 8% SDS-polyacrylamide gel, separated, and transferred onto a nitrocellulose membrane. Quantitative Real-time Reverse Transcription-PCR—Total RNA from cells grown to subconfluency was prepared using the Absolutely RNA reverse transcription-PCR miniprep Kit (Stratagene) according to the manufacturer’s instructions. Determination of Free Cholesterol—Free unesterified cholesterol species were analyzed using the Amplex Red cholesterol assay kit (Molecular Probes) following the manufacturer’s instructions. For determination of the cholesterol content in fractions of sucrose gradients for DRM isolation, 50 ␮l of each fraction were analyzed.

RESULTS

DISCUSSION

ADDITIONS AND CORRECTIONS

Additions and Corrections