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Intracellular metalloprotease activity controls intraneuronal A? aggregation and limits secretion of A? via exosomes.

ABSTRACT: Accumulating evidence suggests that the abnormal aggregation of amyloid-? (??) peptide in Alzheimer's disease (AD) begins intraneuronally, within vesicles of the endosomal-lysosomal pathway where A? is both generated and degraded. Metalloproteases, including endothelin-converting enzyme (ECE)-1 and -2, reside within these vesicles and normally limit the accumulation of intraneuronally produced A?. In this study, we determined whether disruption of A? catabolism could trigger A? aggregation within neurons and increase the amount of A? associated with exosomes, small extracellular vesicles derived from endosomal multivesicular bodies. Using cultured cell lines, primary neurons, and organotypic brain slices from an AD mouse model, we found that pharmacological inhibition of the ECE family of metalloproteases increased intracellular and extracellular A? levels and promoted the intracellular formation of A? oligomers, a process that did not require internalization of secreted A?. In vivo, the accumulation of intraneuronal A? aggregates was accompanied by increased levels of both extracellular and exosome-associated A?, including oligomeric species. Neuronal exosomes were found to contain both ECE-1 and -2 activities, suggesting that multivesicular bodies are intracellular sites of A? degradation by these enzymes. ECE dysfunction could lead to the accumulation of intraneuronal A? aggregates and their subsequent release into the extracellular space via exosomes.-Pacheco-Quinto, J., Clausen, D., Pérez-González, R., Peng, H., Meszaros, A., Eckman, C. B., Levy, E., Eckman, E. A. Intracellular metalloprotease activity controls intraneuronal A? aggregation and limits secretion of A? via exosomes.

SUBMITTER: Pacheco-Quinto J

PROVIDER: S-EPMC6404562 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Intracellular metalloprotease activity controls intraneuronal Aβ aggregation and limits secretion of Aβ via exosomes.

Pacheco-Quinto Javier J Clausen Dana D Pérez-González Rocío R Peng Hui H Meszaros Austin A Eckman Christopher B CB Levy Efrat E Eckman Elizabeth A EA

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20181127 3

Accumulating evidence suggests that the abnormal aggregation of amyloid-β (Αβ) peptide in Alzheimer's disease (AD) begins intraneuronally, within vesicles of the endosomal-lysosomal pathway where Aβ is both generated and degraded. Metalloproteases, including endothelin-converting enzyme (ECE)-1 and -2, reside within these vesicles and normally limit the accumulation of intraneuronally produced Aβ. In this study, we determined whether disruption of Aβ catabolism could trigger Aβ aggregation withi ...[more]

PMID: 30481490

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Project description:BackgroundThe form(s) of amyloid-β peptide (Aβ) associated with the pathology characteristic of Alzheimer's disease (AD) remains unclear. In particular, the neurotoxicity of intraneuronal Aβ accumulation is an issue of considerable controversy; even the existence of Aβ deposits within neurons has recently been challenged by Winton and co-workers. These authors purport that it is actually intraneuronal APP that is being detected by antibodies thought to be specific for Aβ. To further address this issue, an anti-Aβ antibody was developed (MOAB-2) that specifically detects Aβ, but not APP. This antibody allows for the further evaluation of the early accumulation of intraneuronal Aβ in transgenic mice with increased levels of human Aβ in 5xFAD and 3xTg mice.ResultsMOAB-2 (mouse IgG2b) is a pan-specific, high-titer antibody to Aβ residues 1-4 as demonstrated by biochemical and immunohistochemical analyses (IHC), particularly compared to 6E10 (a commonly used commercial antibody to Aβ residues 3-8). MOAB-2 did not detect APP or APP-CTFs in cell culture media/lysates (HEK-APPSwe or HEK-APPSwe/BACE1) or in brain homogenates from transgenic mice expressing 5 familial AD (FAD) mutation (5xFAD mice). Using IHC on 5xFAD brain tissue, MOAB-2 immunoreactivity co-localized with C-terminal antibodies specific for Aβ40 and Aβ42. MOAB-2 did not co-localize with either N- or C-terminal antibodies to APP. In addition, no MOAB-2-immunoreactivity was observed in the brains of 5xFAD/BACE-/- mice, although significant amounts of APP were detected by N- and C-terminal antibodies to APP, as well as by 6E10. In both 5xFAD and 3xTg mouse brain tissue, MOAB-2 co-localized with cathepsin-D, a marker for acidic organelles, further evidence for intraneuronal Aβ, distinct from Aβ associated with the cell membrane. MOAB-2 demonstrated strong intraneuronal and extra-cellular immunoreactivity in 5xFAD and 3xTg mouse brain tissues.ConclusionsBoth intraneuronal Aβ accumulation and extracellular Aβ deposition was demonstrated in 5xFAD mice and 3xTg mice with MOAB-2, an antibody that will help differentiate intracellular Aβ from APP. However, further investigation is required to determine whether a molecular mechanism links the presence of intraneuronal Aβ with neurotoxicity. As well, understanding the relevance of these observations to human AD patients is critical.

Dataset Information

Intracellular metalloprotease activity controls intraneuronal A? aggregation and limits secretion of A? via exosomes.

Publications

Intracellular metalloprotease activity controls intraneuronal Aβ aggregation and limits secretion of Aβ via exosomes.

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