Elevation of β-Amyloid Peptide 2–42 in Sporadic and Familial Alzheimer's Disease and Its Generation in PS1 Knockout Cells

Jens Wiltfang,Hermann Esselmann,Philippe Cupers,Hans Kretzschmar,Johannes Kornhuber,Michael Beyermann,Manuela Neumann,Bart De Strooper,Paul Saftig,Holger Jahn,Eckart Rüther,Detlev Schleuder,Wim Annaert

doi:10.1074/jbc.m102790200

Abstract

Urea-based beta-amyloid (Abeta) SDS-polyacrylamide gel electrophoresis and immunoblots were used to analyze the generation of Abeta peptides in conditioned medium from primary mouse neurons and a neuroglioma cell line, as well as in human cerebrospinal fluid. A comparable and highly conserved pattern of Abeta peptides, namely, 1-40/42 and carboxyl-terminal-truncated 1-37, 1-38, and 1-39, was found. Besides Abeta1-42, we also observed a consistent elevation of amino-terminal-truncated Abeta2-42 in a detergent-soluble pool in brains of subjects with Alzheimer's disease. Abeta2-42 was also specifically elevated in cerebrospinal fluid samples of Alzheimer's disease patients. To decipher the contribution of potential different gamma-secretases (presenilins (PSs)) in generating the amino-terminal- and carboxyl-terminal-truncated Abeta peptides, we overexpressed beta-amyloid precursor protein (APP)-trafficking mutants in PS1+/+ and PS1-/- neurons. As compared with APP-WT (primary neurons from control or PS1-deficient mice infected with Semliki Forest virus), PS1-/- neurons and PS1+/+ neurons overexpressing APP-Deltact (a slow-internalizing mutant) show a decrease of all secreted Abeta peptide species, as expected, because this mutant is processed mainly by alpha-secretase. This drop is even more pronounced for the APP-KK construct (APP mutant carrying an endoplasmic reticulum retention motif). Surprisingly, Abeta2-42 is significantly less affected in PS1-/- neurons and in neurons transfected with the endocytosis-deficient APP-Deltact construct. Our data confirm that PS1 is closely involved in the production of Abeta1-40/42 and the carboxyl-terminal-truncated Abeta1-37, Abeta1-38, and Abeta1-39, but the amino-terminal-truncated and carboxyl-terminal-elongated Abeta2-42 seems to be less affected by PS1 deficiency. Moreover, our results indicate that the latter Abeta peptide species could be generated by a beta(Asp/Ala)-secretase activity.

Highlights

Urea-based ␤-amyloid (A␤) SDS-polyacrylamide gel electrophoresis and immunoblots were used to analyze the generation of A␤ peptides in conditioned medium from primary mouse neurons and a neuroglioma cell line, as well as in human cerebrospinal fluid
Proteolytic cleavages of the integral membrane protein ␤-amyloid precursor protein (APP)1 result in generation of ␤-amyloid (A␤) peptides that accumulate to high levels in the brains of patients suffering from Alzheimer’s disease (AD) [1]
Even if the ␥-secretase has not yet been formally identified, most of the existing in vivo and in vitro data based on presenilin (PS) knockout analysis, mutagenesis of specific amino acids, and drug targeting point to the narrow relationship between presenilins and an aspartyl ␥-secretase activity [15,16,17,18,19,20,21,22,23,24,25]

Summary

Introduction

Urea-based ␤-amyloid (A␤) SDS-polyacrylamide gel electrophoresis and immunoblots were used to analyze the generation of A␤ peptides in conditioned medium from primary mouse neurons and a neuroglioma cell line, as well as in human cerebrospinal fluid. To decipher the contribution of potential different ␥-secretases (presenilins (PSs)) in generating the amino-terminal- and carboxyl-terminaltruncated A␤ peptides, we overexpressed ␤-amyloid precursor protein (APP)-trafficking mutants in PS1؉/؉ and PS1؊/؊ neurons. Proteolytic cleavages of the integral membrane protein ␤-amyloid precursor protein (APP) result in generation of ␤-amyloid (A␤) peptides that accumulate to high levels in the brains of patients suffering from Alzheimer’s disease (AD) [1]. In this regard, one recent notable achievement has been the identification of the ␤-secretase ␤-site amyloid-cleaving enzyme (BACE), a metalloproteinase of 50 kDa [2,3,4,5]. There is some evidence that the carboxyl termini of A␤1– 40 and A␤1– 42 are generated in different cellular compartments [39, 40]

Methods

Results

Conclusion