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A cellular model of amyloid precursor protein processing and amyloid-? peptide production.

ABSTRACT: A hallmark pathologic feature of Alzheimer's disease (AD) is accumulation of neuritic senile plaques in the brain parenchyma. Neurotoxic plaque cores are composed predominantly of amyloid-? (A?) peptides of 40 and 42 amino acids in length, formed by sequential cleavage of amyloid precursor protein (APP) by ?-, and ?-secretases. There is a great interest in approaches to modulate A? peptide production and develop therapeutic interventions to reduce A? levels to halt or slow the progression of neurodegeneration.We characterized and present the BE(2)-M17 human neuroblastoma cell line as a novel in vitro model of the APP-cleavage cascade to support future (1) functional studies of molecular regulators in A? production, and (2) high-throughput screening assays of new pharmacotherapeutics.In BE(2)-M17 cells, both RNA (i.e., RT-PCR, RNA sequencing) and protein analyses (i.e., Western blots, ELISA), show endogenous expression of critical components of the amyloidogenic pathway, APP-cleavage intermediates CTF83 and CTF99, and final cleavage products A?40 and A?42. We further report effects of retinoic acid-mediated differentiation on morphology and gene expression in this cell line.In contrast to primary isolates or other cell lines reported in current literature, BE(2)-M17 not only sustains baseline expression of the full contingent of APP-processing components, but also remains stably adherent during culture, facilitating experimental manipulations.Our evidence supports the use of BE(2)-M17 as a novel, human, cell-based model of the APP processing pathway that offers a potential streamlined approach to dissect molecular functions of endogenous regulatory pathways, and perform mechanistic studies to identify modulators of A? production.

SUBMITTER: Macias MP

PROVIDER: S-EPMC3931259 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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A cellular model of amyloid precursor protein processing and amyloid-β peptide production.

Macias Mimi P MP Gonzales Amanda M AM Siniard Ashley L AL Walker Aaron W AW Corneveaux Jason J JJ Huentelman Matthew J MJ Sabbagh Marwan N MN Decourt Boris B

Journal of neuroscience methods 20131212

<h4>Background</h4>A hallmark pathologic feature of Alzheimer's disease (AD) is accumulation of neuritic senile plaques in the brain parenchyma. Neurotoxic plaque cores are composed predominantly of amyloid-β (Aβ) peptides of 40 and 42 amino acids in length, formed by sequential cleavage of amyloid precursor protein (APP) by β-, and γ-secretases. There is a great interest in approaches to modulate Aβ peptide production and develop therapeutic interventions to reduce Aβ levels to halt or slow the ...[more]

PMID: 24333289

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Project description:The amyloid cascade hypothesis proposes that excessive accumulation of amyloid beta-peptides is the initiating event in Alzheimer's disease. These neurotoxic peptides are generated from the amyloid precursor protein via sequential cleavage by β- and γ-secretases in the 'amyloidogenic' proteolytic pathway. Alternatively, the amyloid precursor protein can be processed via the 'non-amyloidogenic' pathway which, through the action of the α-secretase a disintegrin and metalloproteinase (ADAM) 10, both precludes amyloid beta-peptide formation and has the additional benefit of generating a neuroprotective soluble amyloid precursor protein fragment, sAPPα. In the current study, we investigated whether the orphan drug, dichloroacetate, could alter amyloid precursor protein proteolysis. In SH-SY5Y neuroblastoma cells, dichloroacetate enhanced sAPPα generation whilst inhibiting β-secretase processing of endogenous amyloid precursor protein and the subsequent generation of amyloid beta-peptides. Over-expression of the amyloid precursor protein partly ablated the effect of dichloroacetate on amyloidogenic and non-amyloidogenic processing whilst over-expression of the β-secretase only ablated the effect on amyloidogenic processing. Similar enhancement of ADAM-mediated amyloid precursor protein processing by dichloroacetate was observed in unrelated cell lines and the effect was not exclusive to the amyloid precursor protein as an ADAM substrate, as indicated by dichloroacetate-enhanced proteolysis of the Notch ligand, Jagged1. Despite altering proteolysis of the amyloid precursor protein, dichloroacetate did not significantly affect the expression/activity of α-, β- or γ-secretases. In conclusion, dichloroacetate can inhibit amyloidogenic and promote non-amyloidogenic proteolysis of the amyloid precursor protein. Given the small size and blood-brain-barrier permeability of the drug, further research into its mechanism of action with respect to APP proteolysis may lead to the development of therapies for slowing the progression of Alzheimer's disease.

Dataset Information

A cellular model of amyloid precursor protein processing and amyloid-? peptide production.

Publications

A cellular model of amyloid precursor protein processing and amyloid-β peptide production.

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