Amyloid-b (Ab) protofibrils are known intermediates of the in vitro Ab aggregation process and the protofibrillogenic Arctic mutation (APPE693G) provides clinical support for a pathogenic role of Ab protofibrils in Alzheimer's disease (AD). To verify their in vivo relevance and to establish a quantitative Ab protofibril immunoassay, Ab conformation dependent monoclonal antibodies were generated. One of these antibodies, mAb158 (IgG2a), was used in a sandwich ELISA to specifically detect picomolar concentrations of Ab protofibrils without interference from Ab monomers or the amyloid precursor protein (APP). The specificity and biological significance of this ELISA was demonstrated using cell cultures and transgenic mouse models expressing human APP containing the Swedish mutation (APPKN670/671ML), or the Swedish and Arctic mutation in combination. The mAb158 sandwich ELISA analysis revealed presence of Ab protofibrils in both cell and animal models, proving that Ab protofibrils are formed not only in vitro, but also in vivo. Furthermore, elevated Ab protofibril levels in the Arctic-Swedish samples emphasize the usefulness of the Arctic mutation as a model of enhanced protofibril formation. This assay provides a novel tool for investigating the role of Ab protofibrils in AD and has the potential of becoming an important diagnostic assay.
Amyloid-beta (Abeta) peptide levels are widely measured by enzyme-linked immunosorbent assay (ELISA) in Alzheimer's disease research. Here, we show that oligomerization of Abeta results in underestimated Abeta ELISA levels. The implications are that comprehensive analysis of soluble Abeta requires either sample pretreatment at denaturing conditions or novel conformation-dependent immunoassays. Our findings might be of relevance for many neurodegenerative disorders in which soluble protein aggregates are the main neurotoxic species.
Oligomeric assemblies of amyloid‐β (Aβ) are suggested to be central in the pathogenesis of Alzheimer’s disease because levels of soluble Aβ correlate much better with the extent of cognitive dysfunctions than do senile plaque counts. Moreover, such Aβ species have been shown to be neurotoxic, to interfere with learned behavior and to inhibit the maintenance of hippocampal long‐term potentiation. The tg‐ArcSwe model (i.e. transgenic mice with the Arctic and Swedish Alzheimer mutations) expresses elevated levels of Aβ protofibrils in the brain, making tg‐ArcSwe a highly suitable model for investigating the pathogenic role of these Aβ assemblies. In the present study, we estimated Aβ protofibril levels in the brain and cerebrospinal fluid of tg‐ArcSwe mice, and also assessed their role with respect to cognitive functions. Protofibril levels, specifically measured with a sandwich ELISA, were found to be elevated in young tg‐ArcSwe mice compared to several transgenic models lacking the Arctic mutation. In aged tg‐ArcSwe mice with considerable plaque deposition, Aβ protofibrils were approximately 50% higher than in younger mice, whereas levels of total Aβ were exponentially increased. Young tg‐ArcSwe mice showed deficits in spatial learning, and individual performances in the Morris water maze were correlated inversely with levels of Aβ protofibrils, but not with total Aβ levels. We conclude that Aβ protofibrils accumulate in an age‐dependent manner in tg‐ArcSwe mice, although to a far lesser extent than total Aβ. Our findings suggest that increased levels of Aβ protofibrils could result in spatial learning impairment.
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.
Component-resolved diagnostics before OIT can help to identify children with lower probability of a successful OIT outcome, as high IgE levels to α-lactalbumin, β-lactoglobulin and casein are associated with lower maintenance dose reached. An increase in the IgG4 concentration to milk components during treatment indicated effective desensitization.
Mutations within the amyloid-b (Ab) domain of the amyloid precursor protein (APP) typically generate hemorrhagic strokes and vascular amyloid angiopathy. In contrast, the Arctic mutation (APP E693G) results in Alzheimer's disease. Little is known about the pathologic mechanisms that result from the Arctic mutation, although increased formation of Ab protofibrils in vitro and intraneuronal Ab aggregates in vivo suggest that early steps in the amyloidogenic pathway are facilitated. Here we show that the Arctic mutation favors proamyloidogenic APP processing by increased b-secretase cleavage, as demonstrated by altered levels of N-and C-terminal APP fragments. Although the Arctic mutation is located close to the a-secretase site, APP harboring the Arctic mutation is not an inferior substrate to a disintegrin and metalloprotease-10, a major a-secretase. Instead, the localization of Arctic APP is altered, with reduced levels at the cell surface making Arctic APP less available for a-secretase cleavage. As a result, the extent and subcellular location of Ab formation is changed, as revealed by increased Ab levels, especially at intracellular locations. Our findings suggest that the unique clinical symptomatology and neuropathology associated with the Arctic mutation, but not with other intra-Ab mutations, could relate to altered APP processing with increased steadystate levels of Arctic Ab, particularly at intracellular locations. Keywords: Alzheimer's disease, amyloid-b peptide, amyloid precursor protein processing, Arctic mutation, intracellular amyloid-b, a-secretase.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.