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Specific recognition of biologically active amyloid-? oligomers by a new surface plasmon resonance-based immunoassay and an in vivo assay in Caenorhabditis elegans.

ABSTRACT: Soluble oligomers of the amyloid-? (A?) peptide play a key role in the pathogenesis of Alzheimer's disease, but their elusive nature makes their detection challenging. Here we describe a novel immunoassay based on surface plasmon resonance (SPR) that specifically recognizes biologically active A? oligomers. As a capturing agent, we immobilized on the sensor chip the monoclonal antibody 4G8, which targets a central hydrophobic region of A?. This SPR assay allows specific recognition of oligomeric intermediates that rapidly appear and disappear during the incubation of synthetic A?(1-42), discriminating them from monomers and higher order aggregates. The species recognized by SPR generate ionic currents in artificial lipid bilayers and inhibit the physiological pharyngeal contractions in Caenorhabditis elegans, a new method for testing the toxic potential of A? oligomers. With these assays we found that the formation of biologically relevant A? oligomers is inhibited by epigallocatechin gallate and increased by the A2V mutation, previously reported to induce early onset dementia. The SPR-based immunoassay provides new opportunities for detection of toxic A? oligomers in biological samples and could be adapted to study misfolding proteins in other neurodegenerative disorders.

SUBMITTER: Stravalaci M

PROVIDER: S-EPMC3431664 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Specific recognition of biologically active amyloid-β oligomers by a new surface plasmon resonance-based immunoassay and an in vivo assay in Caenorhabditis elegans.

Stravalaci Matteo M Bastone Antonio A Beeg Marten M Cagnotto Alfredo A Colombo Laura L Di Fede Giuseppe G Tagliavini Fabrizio F Cantù Laura L Del Favero Elena E Del Favero Elena E Mazzanti Michele M Chiesa Roberto R Salmona Mario M Diomede Luisa L Gobbi Marco M

The Journal of biological chemistry 20120626 33

Soluble oligomers of the amyloid-β (Aβ) peptide play a key role in the pathogenesis of Alzheimer's disease, but their elusive nature makes their detection challenging. Here we describe a novel immunoassay based on surface plasmon resonance (SPR) that specifically recognizes biologically active Aβ oligomers. As a capturing agent, we immobilized on the sensor chip the monoclonal antibody 4G8, which targets a central hydrophobic region of Aβ. This SPR assay allows specific recognition of oligomeric ...[more]

PMID: 22736768

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Project description:BackgroundAn essential stage of neural development involves the assembly of neural circuits via formation of inter-neuronal connections. Early steps in neural circuit formation, including cell migration, axon guidance, and the localization of synaptic components, are well described. However, upon reaching their target region, most neurites still contact many potential partners. In order to assemble functional circuits, it is critical that within this group of cells, neurons identify and form connections only with their appropriate partners, a process we call synaptic partner recognition (SPR). To understand how SPR is mediated, we previously developed a genetically encoded fluorescent trans-synaptic marker called NLG-1 GRASP, which labels synaptic contacts between individual neurons of interest in dense cellular environments in the genetic model organism Caenorhabditis elegans.ResultsHere, we describe the first use of NLG-1 GRASP technology, to identify SPR genes that function in this critical process. The NLG-1 GRASP system allows us to assess synaptogenesis between PHB sensory neurons and AVA interneurons instantly in live animals, making genetic analysis feasible. Additionally, we employ a behavioral assay to specifically test PHB sensory circuit function. Utilizing this approach, we reveal a new role for the secreted UNC-6/Netrin ligand and its transmembrane receptor UNC-40/Deleted in colorectal cancer (DCC) in SPR. Synapses between PHB and AVA are severely reduced in unc-6 and unc-40 animals despite normal axon guidance and subcellular localization of synaptic components. Additionally, behavioral defects indicate a complete disruption of PHB circuit function in unc-40 mutants. Our data indicate that UNC-40 and UNC-6 function in PHB and AVA, respectively, to specify SPR. Strikingly, overexpression of UNC-6 in postsynaptic neurons is sufficient to promote increased PHB-AVA synaptogenesis and to potentiate the behavioral response beyond wild-type levels. Furthermore, an artificially membrane-tethered UNC-6 expressed in the postsynaptic neurons promotes SPR, consistent with a short-range signal between adjacent synaptic partners.ConclusionsThese results indicate that the conserved UNC-6/Netrin-UNC-40/DCC ligand-receptor pair has a previously unknown function, acting in a juxtacrine manner to specify recognition of individual postsynaptic neurons. Furthermore, they illustrate the potential of this new approach, combining NLG-1 GRASP and behavioral analysis, in gene discovery and characterization.

Dataset Information

Specific recognition of biologically active amyloid-? oligomers by a new surface plasmon resonance-based immunoassay and an in vivo assay in Caenorhabditis elegans.

Publications

Specific recognition of biologically active amyloid-β oligomers by a new surface plasmon resonance-based immunoassay and an in vivo assay in Caenorhabditis elegans.

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