Article

Pathogenic APP mutations near the gamma-secretase cleavage site differentially affect Abeta secretion and APP C-terminal fragment stability.

Laboratory of Neurogenetics, Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology, University of Antwerp, Belgium.
Human Molecular Genetics (Impact Factor: 6.68). 09/2001; 10(16):1665-71.
Source: PubMed

ABSTRACT Release of amyloid beta (Abeta) from the amyloid precursor protein (APP) requires cleavages by beta- and gamma-secretases and plays a crucial role in Alzheimer's disease (AD) pathogenesis. Missense mutations in the APP gene causing familial AD are clustered around the beta-, alpha- and particular gamma-secretase cleavage sites. We systematically compare in primary neurons the effect on APP processing of a series of clinical APP mutations (two of which not characterized before) located in close proximity to the gamma-secretase cleavage site. We confirm and extend previous observations showing that all these mutations (T714I, V715M, V715A, I716V, V717I and V717L) affect gamma-secretase cleavage causing an increased relative ratio of Abeta42 to Abeta40. Taking advantage of these extended series of APP mutations we were able to demonstrate an inverse correlation between these ratios and the age at onset of the disease in the different families. In addition, a subset of mutations caused the accumulation of APP C-terminal fragments indicating that these mutations also influence the stability of APP C-terminal fragments. However, it is unlikely that these fragments contribute significantly to the disease process.

0 Bookmarks
 · 
63 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Pathogenic mutations in the Amyloid precursor protein (APP) gene have been described as causing early onset familial Alzheimer's disease (AD). We recently identified a rare APP variant encoding an alanine-to-threonine substitution at residue 673 (A673T) that confers protection against development of AD (1). The A673 residue lies within the β-secretase recognition sequence, and is part of the Aβ peptide cleavage product (position 2 of Aβ). We previously demonstrated that the A673T substitution makes APP a less favorable substrate for cleavage by BACE1. In follow-up studies, we confirm that A673T APP shows reduced cleavage by BACE1 in transfected mouse primary neurons and in isogenic human iPSC-derived neurons. Using a biochemical approach, we show that the A673T substitution modulates the catalytic turnover rate (Vmax) of APP by the BACE1 enzyme, without affecting the affinity (Km) of the APP substrate for BACE1. We also show a reduced level of Aβ1-42 aggregation with A2T Aβ peptides, an observation not conserved in Aβ1-40 peptides. When combined in a ratio of 1:9 Aβ1-42:Aβ1-40 to mimic physiologically relevant mixtures, A2T retains a trend toward slowed aggregation kinetics. Microglial uptake of the mutant Aβ1-42 peptides correlated with their aggregation level. Cytotoxicity of the mutant Aβ peptides was not dramatically altered. Taken together, our findings demonstrate that A673T, a protective allele of APP, reproducibly reduces amyloidogenic processing of APP and also mildly decreases Aβ aggregation. These effects could together have an additive or even synergistic impact on risk of developing AD.
    Journal of Biological Chemistry 09/2014; DOI:10.1074/jbc.M114.589069 · 4.60 Impact Factor
  • Source
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Alzheimer's disease (AD), a progressive neurodegenerative disorder that is the most common cause of dementia in the elderly, is characterized by the accumulation of amyloid-beta (Abeta) plaques and neurofibrillary tangles, as well as a progressive loss of synapses and neurons in the brain. The major pertinacious component of amyloid plaques is Abeta, a variably sized peptide derived from the integral membrane protein amyloid precursor protein (APP). The Abeta region of APP locates partly within its ecto- and trans-membrane domains. APP is cleaved by three proteases, designated as alpha-, beta-, and gamma-secretases. Processing by beta- and gamma-secretase cleaves the N- and C-terminal ends of the Abeta region, respectively, releasing Abeta, whereas alpha-secretase cleaves within the Abeta sequence, releasing soluble APPalpha (sAPPalpha). The gamma-secretase cleaves at several adjacent sites to yield Abeta species containing 39-43 amino acid residues. Both alpha- and beta-cleavage sites of human wild-type APP are located in APP672-699 region (ectodomain of beta-C-terminal fragment, ED-beta-CTF or ED-C99). Therefore, the amino acid residues within or near this region are definitely pivotal for human wild-type APP function and processing. Here, we report that one ED-C99-specific monoclonal antibody (mAbED-C99) blocks human wild-type APP endocytosis and shifts its processing from alpha- to beta-cleavage, as evidenced by elevated accumulation of cell surface full-length APP and beta-CTF together with reduced sAPPalpha and alpha-CTF levels. Moreover, mAbED-C99 enhances the interactions of APP with cholesterol. Consistently, intracerebroventricular injection of mAbED-C99 to human wild-type APP transgenic mice markedly increases membrane-associated beta-CTF. All these findings suggest that APP672-699 region is critical for human wild-type APP processing and may provide new clues for the pathogenesis of sporadic AD.
    Cell Death & Disease 08/2014; 5:e1374. DOI:10.1038/cddis.2014.336 · 5.18 Impact Factor