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Constitutive ?- and ?-secretase cleavages of the amyloid precursor protein are partially coupled in neurons, but not in frequently used cell lines.

ABSTRACT: Proteolytic cleavage of the amyloid precursor protein (APP) by the two proteases ?- and ?-secretases controls the generation of the amyloid ? peptide (A?), a key player in Alzheimer's disease pathogenesis. The ?-secretase ADAM10 and the ?-secretase BACE1 have opposite effects on A? generation and are assumed to compete for APP as a substrate, such that their cleavages are inversely coupled. This concept was mainly demonstrated in studies using activation or overexpression of ?- and ?-secretases. Here, we report that this inverse coupling is not seen to the same extent upon inhibition of the endogenous proteases. Genetic and pharmacological inhibition of ADAM10 and BACE1 revealed that the endogenous, constitutive ?-secretase cleavage of APP is largely uncoupled from ?-secretase cleavage and A? generation in neuroglioma H4 cells and in neuronally differentiated SH-SY5Y cells. In contrast, inverse coupling was observed in primary cortical neurons. However, this coupling was not bidirectional. Inhibition of BACE1 increased ADAM10 cleavage of APP, but a reduction of ADAM10 activity did not increase the BACE1 cleavage of APP in the neurons. Our analysis shows that the inverse coupling of the endogenous ?- and ?-secretase cleavages depends on the cellular model and suggests that a reduction of ADAM10 activity is unlikely to increase the AD risk through increased ?-secretase cleavage.

SUBMITTER: Colombo A

PROVIDER: S-EPMC4310234 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Constitutive α- and β-secretase cleavages of the amyloid precursor protein are partially coupled in neurons, but not in frequently used cell lines.

Colombo Alessio A Wang Huanhuan H Kuhn Peer-Hendrik PH Page Richard R Kremmer Elisabeth E Dempsey Peter J PJ Crawford Howard C HC Lichtenthaler Stefan F SF

Neurobiology of disease 20120824

Proteolytic cleavage of the amyloid precursor protein (APP) by the two proteases α- and β-secretases controls the generation of the amyloid β peptide (Aβ), a key player in Alzheimer's disease pathogenesis. The α-secretase ADAM10 and the β-secretase BACE1 have opposite effects on Aβ generation and are assumed to compete for APP as a substrate, such that their cleavages are inversely coupled. This concept was mainly demonstrated in studies using activation or overexpression of α- and β-secretases. ...[more]

PMID: 22940630

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Project description:INTRODUCTION: Inhibition of gamma-secretase presents a direct target for lowering A? production in the brain as a therapy for Alzheimer's disease (AD). However, gamma-secretase is known to process multiple substrates in addition to amyloid precursor protein (APP), most notably Notch, which has limited clinical development of inhibitors targeting this enzyme. It has been postulated that APP substrate selective inhibitors of gamma-secretase would be preferable to non-selective inhibitors from a safety perspective for AD therapy. METHODS: In vitro assays monitoring inhibitor potencies at APP ?-site cleavage (equivalent to A?40), and Notch ?-site cleavage, in conjunction with a single cell assay to simultaneously monitor selectivity for inhibition of A? production vs. Notch signaling were developed to discover APP selective gamma-secretase inhibitors. In vivo efficacy for acute reduction of brain A? was determined in the PDAPP transgene model of AD, as well as in wild-type FVB strain mice. In vivo selectivity was determined following seven days x twice per day (b.i.d.) treatment with 15 mg/kg/dose to 1,000 mg/kg/dose ELN475516, and monitoring brain A? reduction vs. Notch signaling endpoints in periphery. RESULTS: The APP selective gamma-secretase inhibitors ELN318463 and ELN475516 reported here behave as classic gamma-secretase inhibitors, demonstrate 75- to 120-fold selectivity for inhibiting A? production compared with Notch signaling in cells, and displace an active site directed inhibitor at very high concentrations only in the presence of substrate. ELN318463 demonstrated discordant efficacy for reduction of brain A? in the PDAPP compared with wild-type FVB, not observed with ELN475516. Improved in vivo safety of ELN475516 was demonstrated in the 7d repeat dose study in wild-type mice, where a 33% reduction of brain A? was observed in mice terminated three hours post last dose at the lowest dose of inhibitor tested. No overt in-life or post-mortem indications of systemic toxicity, nor RNA and histological end-points indicative of toxicity attributable to inhibition of Notch signaling were observed at any dose tested. CONCLUSIONS: The discordant in vivo activity of ELN318463 suggests that the potency of gamma-secretase inhibitors in AD transgenic mice should be corroborated in wild-type mice. The discovery of ELN475516 demonstrates that it is possible to develop APP selective gamma-secretase inhibitors with potential for treatment for AD.

Dataset Information

Constitutive ?- and ?-secretase cleavages of the amyloid precursor protein are partially coupled in neurons, but not in frequently used cell lines.

Publications

Constitutive α- and β-secretase cleavages of the amyloid precursor protein are partially coupled in neurons, but not in frequently used cell lines.

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