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The generation of the amyloid-β (Aβ) peptides from the amyloid precursor protein (APP) through sequential proteolysis by β- and γ-secretases is a key pathological event in the initiation and propagation of Alzheimer's disease. Aβ and the transcriptionally active APP intracellular domain are generated preferentially from the APP695 isoform compared to the longer APP751 isoform. As the Aβ and amyloid precursor protein intracellular domain produced from cleavage of APP695 and APP751 are identical we hypothesised that the two isoforms have differences within their interactomes which mediate the differential processing of the two isoforms. To investigate this, we applied a proteomics-based approach to identify differences in the interactomes of the APP695 and APP751 isoforms. Using stable isotope labelling of amino acids in cell culture and quantitative proteomics, we compared the interactomes of APP695 and APP751 expressed in human SH-SY5Y cells. Through this approach, we identified enrichment of proteins involved in mitochondrial function, the nuclear pore and nuclear transport specifically in the APP695 interactome. Further interrogation of the APP interactome and subsequent experimental validation (co-immunoprecipitation and siRNA knockdown) revealed GAP43 as a specific modulator of APP751 proteolysis, altering Aβ generation. Our data indicate that interrogation of the APP interactome can be exploited to identify proteins which influence APP proteolysis and Aβ production in an isoform dependent-manner. Cover Image for this issue: doi: 10.1111/jnc.14504., (© 2019 International Society for Neurochemistry.)

Proteolysis of the amyloid precursor protein (APP) liberates various fragments including the proposed initiator of Alzheimer disease-associated dysfunctions, amyloid-β. However, recent evidence suggests that the accepted view of APP proteolysis by the canonical α-, β-, and γ-secretases is simplistic, with the discovery of a number of novel APP secretases (including δ- and η-secretases, alternative β-secretases) and additional metabolites, some of which may also cause synaptic dysfunction. Furthermore, various proteins have been identified that interact with APP and modulate its cleavage by the secretases. Here, we give an overview of the increasingly complex picture of APP proteolysis., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The cellular prion protein (PrPC) has been proposed to play an important role in the pathogenesis of Alzheimer's disease. In cellular models PrPC inhibited the action of the β-secretase BACE1 on wild type amyloid precursor protein resulting in a reduction in amyloid-β (Aβ) peptides. Here we have assessed the effect of genetic ablation of PrPC in transgenic mice expressing human wild type amyloid precursor protein (line I5). Deletion of PrPC had no effect on the α- and β-secretase proteolysis of the amyloid precursor protein (APP) nor on the amount of Aβ38, Aβ40 or Aβ42 in the brains of the mice. In addition, ablation of PrPC did not alter Aβ deposition or histopathology phenotype in this transgenic model. Thus using this transgenic model we could not provide evidence to support the hypothesis that PrPC regulates Aβ production.

In Alzheimer disease amyloid-β (Aβ) peptides derived from the amyloid precursor protein (APP) accumulate in the brain. Cleavage of APP by the β-secretase BACE1 is the rate-limiting step in the production of Aβ. We have reported previously that the cellular prion protein (PrP(C)) inhibited the action of BACE1 toward human wild type APP (APP(WT)) in cellular models and that the levels of endogenous murine Aβ were significantly increased in PrP(C)-null mouse brain. Here we investigated the molecular and cellular mechanisms underlying this observation. PrP(C) interacted directly with the prodomain of the immature Golgi-localized form of BACE1. This interaction decreased BACE1 at the cell surface and in endosomes where it preferentially cleaves APP(WT) but increased it in the Golgi where it preferentially cleaves APP with the Swedish mutation (APP(Swe)). In transgenic mice expressing human APP with the Swedish and Indiana familial mutations (APP(Swe,Ind)), PrP(C) deletion had no influence on APP proteolytic processing, Aβ plaque deposition, or levels of soluble Aβ or Aβ oligomers. In cells, although PrP(C) inhibited the action of BACE1 on APP(WT), it did not inhibit BACE1 activity toward APP(Swe). The differential subcellular location of the BACE1 cleavage of APP(Swe) relative to APP(WT) provides an explanation for the failure of PrP(C) deletion to affect Aβ accumulation in APP(Swe,Ind) mice. Thus, although PrP(C) exerts no control on cleavage of APP(Swe) by BACE1, it has a profound influence on the cleavage of APP(WT), suggesting that PrP(C) may be a key protective player against sporadic Alzheimer disease.

Amyloidogenic processing of the amyloid precursor protein (APP) by β- and γ-secretases generates several biologically active products, including amyloid-β (Aβ) and the APP intracellular domain (AICD). AICD regulates transcription of several neuronal genes, especially the Aβ-degrading enzyme, neprilysin (NEP). APP exists in several alternatively spliced isoforms, APP(695), APP(751), and APP(770). We have examined whether each isoform can contribute to AICD generation and hence up-regulation of NEP expression. Using SH-SY5Y neuronal cells stably expressing each of the APP isoforms, we observed that only APP(695) up-regulated nuclear AICD levels (9-fold) and NEP expression (6-fold). Increased NEP expression was abolished by a β- or γ-secretase inhibitor but not an α-secretase inhibitor. This correlated with a marked increase in both Aβ(1-40) and Aβ(1-42) in APP(695) cells as compared with APP(751) or APP(770) cells. Similar phenomena were observed in Neuro2a but not HEK293 cells. SH-SY5Y cells expressing the Swedish mutant of APP(695) also showed an increase in Aβ levels and NEP expression as compared with wild-type APP(695) cells. Chromatin immunoprecipitation revealed that AICD was associated with the NEP promoter in APP(695), Neuro2a, and APP(Swe) cells but not APP(751) nor APP(770) cells where AICD was replaced by histone deacetylase 1 (HDAC1). AICD occupancy of the NEP promoter was replaced by HDAC1 after treatment of the APP(695) cells with a β- but not an α-secretase inhibitor. The increased AICD and NEP levels were significantly reduced in cholesterol-depleted APP(695) cells. In conclusion, Aβ and functional AICD appear to be preferentially synthesized through β-secretase action on APP(695).

Bridging integrator 1 (BIN1) has been implicated in sporadic Alzheimer’s disease (AD) by a number of genome wide association studies (GWAS) in a variety of populations. Here we measured BIN1 in frontal cortex samples from 24 sporadic AD and 24 age-matched non-dementia brains and correlated the expression of this protein with markers of AD. BIN1 was reduced by 87% (p=0.007) in sporadic AD compared to non-dementia controls, but BIN1 in sporadic AD did not correlate with soluble Aβ (rs=-0.084, p=0.698), insoluble Aβ (rs=0.237, p=0.269), Aβ plaque load (rs=0.063, p=0.771) or phospho-tau load (rs=-0.160, p=0.489). In contrast to our findings in sporadic AD, BIN1 was unchanged in the hippocampus from 6 cases of familial AD compared to 6 age-matched controls (p=0.488). BIN1 declined with age in a cohort of non-dementia control cases between 25 and 88 years but the correlation was not significant (rs=-0.449, p=0.081). Although BIN1 is known to have a role in endocytosis, and the processing of the amyloid precursor protein (APP) to form amyloid-β (Aβ) peptides is dependent on endocytosis, knockdown of BIN1 by targeted siRNA or the overexpression of BIN1 in a human neuroblastoma cell line (SH-SY5Y) had no effect on APP processing. These data suggest that the alteration in BIN1 is involved in the pathogenesis of sporadic, but not familial AD and is not a consequence of AD neurodegeneration or the ageing process, a finding in keeping with the numerous GWAS that implicate BIN1 in sporadic AD. However, the mechanism of its contribution remains to be established. [ABSTRACT FROM AUTHOR]