Alzheimer amyloid peptide aß42 regulates gene expression of transcription and growth factors.

The pathogenesis of Alzheimer's disease (AD) is characterized by the aggregation of amyloid-ß (Aß) peptides leading to deposition of senile plaques and a progressive decline of cognitive functions, which currently remains the main criterion for its diagnosis. Robust biomarkers for AD do not yet exist, although changes in the cerebrospinal fluid levels of tau and Aß represent promising candidates in addition to brain imaging and genetic risk profiling. Although concentrations of soluble Aß42 correlate with symptoms of AD, less is known about the biological activities of Aß peptides which are generated from the amyloid-ß protein precursor. An unbiased DNA microarray study showed that Aß42, at sub-lethal concentrations, specifically increases expression of several genes in neuroblastoma cells, notably the insulin-like growth factor binding proteins 3 and 5 (IGFBP3/5), the transcription regulator inhibitor of DNA binding, and the transcription factor Lim only domain protein 4. Using qRT-PCR, we confirmed that mRNA levels of the identified candidate genes were exclusively increased by the potentially neurotoxic Aß42 wild-type peptide, as both the less toxic Aß40 and a non-toxic substitution peptide Aß42 G33A did not affect mRNA levels. In vivo immunohistochemistry revealed a corresponding increase in both hippocampal and cortical IGFBP5 expression in an AD mouse model. Proteomic analyses of human AD cerebrospinal fluid displayed increased in vivo concentrations of IGFBPs. IGFBPs and transcription factors, as identified here, are modulated by soluble Aß42 and may represent useful early biomarkers.

Characterization of intermediate steps in amyloid beta (Aß) production under near-native conditions.

Processing of the amyloid precursor protein (APP) by γ-secretase results in generation of Aß peptides of different lengths ranging from 51 to 30 residues. Accumulation of Aß and in particular Aß42 is enhanced by familial Alzheimer disease (FAD) causing mutations in APP and is believed to play a pivotal role. The molecular mechanism underlying normal Aß production, the impact of FAD mutations on this process and how anti-amyloidogenic γ-secretase modulators (GSMs) cause a selective decrease in Aß40 and Aß42 and an increase in shorter Aß peptides, however, is poorly understood. By using a combined immuno- and LC-MS-based assay we identify several major intermediates, i.e. 3- and 4-peptides that line up head to head across the entire APP transmembrane sequence from Aß51 to Aß31/Aß30 and from Aß49 to Aß30/31. FAD APP mutations displayed a relative increase in 3- and 4-peptides from Aß48 to Aß38 compared with Aß49 to Aß37. These findings correlate with an increase in the Aß42/40 ratio. GSMs caused a decrease in Aß40 and Aß42 and an increase in Aß37 and Aß38 paralleled by an increase of the intermediates Aß40-38 and Aß42-39. Collectively, these data provide a thorough characterization of all intermediate steps in Aß production in native cell membranes and provide key mechanistic insights to genetic and pharmacological modulation of Aß generation.

Novel APP/Aß mutation K16N produces highly toxic heteromeric Aß oligomers.

Here, we describe a novel missense mutation in the amyloid precursor protein (APP) causing a lysine-to-asparagine substitution at position 687 (APP770; herein, referred to as K16N according to amyloid-ß (Aß) numbering) resulting in an early onset dementia with an autosomal dominant inheritance pattern. The K16N mutation is located exactly at the α-secretase cleavage site and influences both APP and Aß. First, due to the K16N mutation APP secretion is affected and a higher amount of Aß peptides is being produced. Second, Aß peptides carrying the K16N mutation are unique in that the peptide itself is not harmful to neuronal cells. Severe toxicity, however, is evident upon equimolar mixture of wt and mutant peptides, mimicking the heterozygous state of the subject. Furthermore, Aß42 K16N inhibits fibril formation of Aß42 wild-type. Even more, Aß42 K16N peptides are protected against clearance activity by the major Aß-degrading enzyme neprilysin. Thus the mutation characterized here harbours a combination of risk factors that synergistically may contribute to the development of early onset Alzheimer disease.

The amyloid precursor protein C-terminal fragment C100 occurs in monomeric and dimeric stable conformations and binds γ-secretase modulators.

The amyloid-ß (Aß) peptide is contained within the C-terminal fragment (ß-CTF) of the amyloid precursor protein (APP) and is intimately linked to Alzheimer's disease. In vivo, Aß is generated by sequential cleavage of ß-CTF within the γ-secretase module. To investigate γ-secretase function, in vitro assays are in widespread use which require a recombinant ß-CTF substrate expressed in bacteria and purified from inclusion bodies, termed C100. So far, little is known about the conformation of C100 under different conditions of purification and refolding. Since C100 dimerization influences the efficiency and specificity of γ-secretase cleavage, it is also of great interest to determine the secondary structure and the oligomeric state of the synthetic substrate as well as the binding properties of small molecules named γ-secretase modulators (GSMs) which we could previously show to modulate APP transmembrane sequence interactions [Richter et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14597-14602]. Here, we use circular dichroism and continuous-wave electron spin resonance measurements to show that C100 purified in a buffer containing SDS at micelle-forming concentrations adopts a highly stable α-helical conformation, in which it shows little tendency to aggregate or to form higher oligomers than dimers. By surface plasmon resonance analysis and molecular modeling we show that the GSM sulindac sulfide binds to C100 and has a preference for C100 dimers.

Aberrant amyloid precursor protein (APP) processing in hereditary forms of Alzheimer disease caused by APP familial Alzheimer disease mutations can be rescued by mutations in the APP GxxxG motif.

The identification of hereditary familial Alzheimer disease (FAD) mutations in the amyloid precursor protein (APP) and presenilin-1 (PS1) corroborated the causative role of amyloid-beta peptides with 42 amino acid residues (Abeta42) in the pathogenesis of AD. Although most FAD mutations are known to increase Abeta42 levels, mutations within the APP GxxxG motif are known to lower Abeta42 levels by attenuating transmembrane sequence dimerization. Here, we show that aberrant Abeta42 levels of FAD mutations can be rescued by GxxxG mutations. The combination of the APP-GxxxG mutation G33A with APP-FAD mutations yielded a constant 60% decrease of Abeta42 levels and a concomitant 3-fold increase of Abeta38 levels compared with the Gly(33) wild-type as determined by ELISA. In the presence of PS1-FAD mutations, the effects of G33A were attenuated, apparently attributable to a different mechanism of PS1-FAD mutants compared with APP-FAD mutants. Our results contribute to a general understanding of the mechanism how APP is processed by the gamma-secretase module and strongly emphasize the potential of the GxxxG motif in the prevention of sporadic AD as well as FAD.