Improved electrophoretic separation and immunoblotting of beta‐amyloid (Aβ) peptides 1–40, 1–42, and 1–43

Abstract

Beta-amyloid peptides (A beta peptides) form the main protein component of the amyloid deposits found in the brains of Alzheimer's disease (AD) patients. Soluble A beta peptides, which are proteolytic fragments of the amyloid-precursor protein (APP) are constitutively secreted by cells expressing APP during normal metabolism [1] and are also present in human plasma and cerebrospinal fluid [2]. Missense mutations in Codon 717 of the APP gene are responsible for a small percentage of inherited AD cases (FAD) and increase the amount of A beta peptides containing additional carboxy terminal amino acids (A beta 1-42, A beta 1-43) [3, 4]. Recent findings indicate that FAD mutations in the presenilin 1 and 2 genes also increase the amount of these longer A beta peptides [5]. A beta 1-42 polymerizes more rapidly in vitro [6] than A beta 1-40 and has been identified as the major component of the brain amyloid deposits [7-9]. We recently developed a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system [10] for the separation of these two peptides. Here we describe a modified version of the original SDS-PAGE procedure, which allows the separation of A beta 1-40, A beta 1-42, and A beta 1-43 for the first time. Detection of the three A beta peptides in the lower ng and pg range is realized by optimized silver staining or immunoblot procedures. These nonradioactive methods may validate results obtained by ELISA procedures used to study the metabolic fate of APP. They may help to define the neurotoxic potential of the longer A beta peptides in relation to their aggregation state.

Presenilin-1 mutations of leucine 166 equally affect the generation of the Notch and APP intracellular domains independent of their effect on Aβ 42 production

“…In contrast, significantly reduced luciferase activity was observed with the L166P and L166R mutants. Thus, these data demonstrate that in addition to producing aberrant amounts of A␤ 42 , the L166P and L166R mutants are also partially defective in Notch signaling.…”

“…Almost all of these mutations cause the increased generation of the highly amyloidogenic 42-aa-long variant of A␤ (A␤ 42 ) that aggregates faster than the shorter A␤ 40 and is predominantly deposited in senile plaques (1). However, by far the highest numbers of mutations linked with FAD have been identified in the PS1 gene.…”

“…additional mutagenesis demonstrate that pathogenic A␤ 42 production and proteolytic generation of AICD or NICD are differentially affected by PS1 L166 mutants. Finally, we show that PS1 L166 mutants affect NICD and AICD generation in a similar manner, suggesting that S3 protease cleavage and the S3-like ␥-secretase cleavage are mediated by the same PSdependent ␥-secretase activity.…”

“…In addition, rare mutations causing FAD have also been found in the homologous PS2 gene. Like most of the FAD-associated APP mutations, FAD mutations in PS1 and PS2 cause the increased generation of A␤ 42 . Interestingly, several FAD-associated PS1 mutations have been shown to impair Notch signaling (22) and consistent with this observation showed reduced rescuing activity of the egg-laying defect of a mutant Caenorhabditis elegans sel-12 PS homologue (23,24).…”

CSF amyloid-β-peptides in Alzheimer's disease, dementia with Lewy bodies and Parkinson's disease dementia

“…For separation of Aβ peptides and subsequent detection, 10 μl of unconcentrated CSF was boiled in a sample buffer for SDS–PAGE, and Aβ-SDS–PAGE/immunoblot was conducted as published elsewhere (Wiltfang et al ., 1997, 2002). …”