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Large Aggregates Are the Major Soluble Ab Species in AD Brain Fractionated with Density Gradient Ultracentrifugation
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. (Molecular Geriatrics)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics. (Molecular Geriatrics)
Uppsala University, Disciplinary Domain of Science and Technology, Technology, Department of Engineering Sciences, Microsystems Technology.
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2012 (English)In: PLoS ONE, ISSN 1932-6203, Vol. 7, no 2, e32014Article in journal (Refereed) Published
Abstract [en]

Soluble amyloid-β (Aβ) aggregates of various sizes, ranging from dimers to large protofibrils, have been associated with neurotoxicity and synaptic dysfunction in Alzheimer's Disease (AD). To investigate the properties of biologically relevant Aβ species, brain extracts from amyloid β protein precursor (AβPP) transgenic mice and AD patients as well as synthetic Aβ preparations were separated by size under native conditions with density gradient ultracentrifugation. The fractionated samples were then analyzed with atomic force microscopy (AFM), ELISA, and MTT cell viability assay. Based on AFM appearance and immunoreactivity to our protofibril selective antibody mAb158, synthetic Aβ42 was divided in four fractions, with large aggregates in fraction 1 and the smallest species in fraction 4. Synthetic Aβ aggregates from fractions 2 and 3 proved to be most toxic in an MTT assay. In AβPP transgenic mouse brain, the most abundant soluble Aβ species were found in fraction 2 and consisted mainly of Aβ40. Also in AD brains, Aβ was mainly found in fraction 2 but primarily as Aβ42. All biologically derived Aβ from fraction 2 was immunologically discriminated from smaller species with mAb158. Thus, the predominant species of biologically derived soluble Aβ, natively separated by density gradient ultracentrifugation, were found to match the size of the neurotoxic, 80–500 kDa synthetic Aβ protofibrils and were equally detected with mAb158.

Place, publisher, year, edition, pages
2012. Vol. 7, no 2, e32014
Keyword [en]
Alzheimer's disease, amyloid-beta, aggregates, protofibrils
National Category
Medical and Health Sciences Engineering and Technology
Research subject
URN: urn:nbn:se:uu:diva-132734DOI: 10.1371/journal.pone.0032014ISI: 000302741300108OAI: oai:DiVA.org:uu-132734DiVA: diva2:359016
Available from: 2010-10-26 Created: 2010-10-26 Last updated: 2015-05-26Bibliographically approved
In thesis
1. Aβ Conformation Dependent Antibodies and Alzheimer's Disease
Open this publication in new window or tab >>Aβ Conformation Dependent Antibodies and Alzheimer's Disease
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Soluble intermediates of the amyloid-β (Aβ) aggregation process are suggested to play a central role in the pathogenesis of Alzheimer’s disease (AD) by causing synaptic dysfunction and neuronal loss. In this thesis, soluble Aβ aggregates have been studied with a particular focus on the Aβ protofibril, which has served as the antigen for developing conformation dependent monoclonal antibodies.

Antibodies generated from mice immunized with Aβ protofibrils were characterized regarding Aβ binding properties and the amino acid sequences of their antigen binding sites. A conformation dependent IgG antibody, mAb158, was further characterized and found to bind to Aβ protofibrils with a 200-fold higher affinity than to monomeric Aβ without affinity for soluble amyloid-β precursor protein (AβPP) or other amyloidogenic proteins. A sandwich enzyme-linked immunosorbent assay (ELISA) based on mAb158 was used to measure soluble Aβ protofibrils in brain extracts from AβPP-transgenic mice. Low levels of protofibrils could also be detected in human AD brain. However, positive signals generated from measurements in AD and control CSF samples were attributed to interference from heterophilic antibodies (HA), generating false positive signals by cross-binding the assay antibodies; consequently, a study on HA interference in Aβ oligomer ELISAs was initiated. A large set of plasma and CSF samples from AD and non-AD subjects were analyzed with and without measures taken to block HA interference, revealing that virtually all signals above the assay limit of detection were false and generated by HA interference.

Many types of soluble Aβ aggregates have been described and suggested to impair neuron and synapse function. To investigate the soluble Aβ pool, synthetic Aβ and brain extracts from AβPP-transgenic mice and AD patients were ultracentrifuged on a density gradient to separate Aβ by size under native conditions. Four distinct gradient fractions were defined based on the appearance of synthetic Aβ in atomic force microscopy (AFM) and immunoreactivity in our protofibril specific sandwich ELISA. Interestingly, most Aβ from AD patients and AβPP-transgenic mice separated in the same fraction as toxic synthetic protofibrils.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 69 p.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 623
Alzheimer's disease, amyloid-beta, protofibrils, conformation, monoclonal antibody, ELISA
National Category
Medical and Health Sciences
Research subject
urn:nbn:se:uu:diva-132736 (URN)978-91-554-7949-7 (ISBN)
Public defence
2010-12-17, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Swedish Research Council, 2004-2167
Available from: 2010-11-25 Created: 2010-10-26 Last updated: 2011-01-13Bibliographically approved

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Publisher's full texthttp://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0032014

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