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Background: Despite the clear physical association between activated astrocytes and amyloid-beta (A beta) plaques, the importance of astrocytes and their therapeutic potential in Alzheimer's disease remain elusive. Soluble A beta aggregates, such as protofibrils, have been suggested to be responsible for the widespread neuronal cell death in Alzheimer's disease, but the mechanisms behind this remain unclear. Moreover, ineffective degradation is of great interest when it comes to the development and progression of neurodegeneration. Based on our previous results that astrocytes are extremely slow in degrading phagocytosed material, we hypothesized that astrocytes may be an important player in these processes. Hence, the aim of this study was to clarify the role of astrocytes in clearance, spreading and neuronal toxicity of A beta. Results: To examine the role of astrocytes in A beta pathology, we added A beta protofibrils to a co-culture system of primary neurons and glia. Our data demonstrates that astrocytes rapidly engulf large amounts of A beta protofibrils, but then store, rather than degrade the ingested material. The incomplete digestion results in a high intracellular load of toxic, partly N-terminally truncated A beta and severe lysosomal dysfunction. Moreover, secretion of microvesicles containing N-terminally truncated A beta, induce apoptosis of cortical neurons. Conclusions: Taken together, our results suggest that astrocytes play a central role in the progression of Alzheimer's disease, by accumulating and spreading toxic A beta species.

The Alzheimer's disease (AD)-related peptide amyloid-beta (A beta) has a propensity to aggregate into various assemblies including toxic soluble A beta protofibrils. Several studies have reported the existence of anti-A beta antibodies in humans. However, it is still debated whether levels of anti-A beta antibodies are altered in AD patients compared to healthy individuals. Formation of immune complexes with plasma A beta makes it difficult to reliably measure the concentration of circulating anti-A beta antibodies with certain immunoassays, potentially leading to an underestimation. Here we have investigated anti-A beta antibody production on a cellular level by measuring the amount of anti-A beta antibody producing cells instead of the plasma level of anti-A beta antibodies. To our knowledge, this is the first time the anti-A beta antibody response in plasma has been compared in AD patients and age-matched healthy individuals using the enzyme-linked immunospot (ELISpot) technique. Both AD patients and healthy individuals had low levels of B cells producing antibodies binding A beta(40) monomers, whereas the number of cells producing antibodies toward A beta(42) protofibrils was higher overall and significantly higher in AD compared to healthy controls. This study shows, by an alternative and reliable method, that there is a specific immune response to the toxic A beta protofibrils, which is significantly increased in AD patients.

Amyloid-beta (Abeta) oligomers of different sizes and forms have recently been the focus for many Alzheimer's disease (AD) researchers. Various immunoassays have been used to detect low concentrations of these elusive Abeta species in different forms of human samples using little or no sample dilutions. However, the possibility that positive results may be caused by interference from heterophilic antibodies (HA) is often overlooked. HA, which recognize immunoglobulins from other species, are present in human plasma and cerebrospinal fluid (CSF) and may cause interference in sandwich immunoassays like enzyme-linked immunosorbent assays (ELISAs) by cross-binding the capture and detection antibodies of the assay. They thus may generate a false positive signal. Here we show that when assessing the Abeta oligomer content in plasma samples from 44 individuals with a sandwich ELISA, none of the 21 positive signals remained when the assay was repeated in the presence of factors blocking HA. Similarly, in CSF samples from 104 individuals, the signals from the 22 positive samples were strongly reduced when analyzed after anti-HA treatment. Taken together, HA interference is a problem that needs to be addressed when measuring low levels of an antigen in human plasma and CSF samples., Title also written as: Interference from Heterophilic Antibodies in Amyloid-β Oligomer ELISAs

Soluble oligomeric aggregates of the amyloid-beta peptide (Abeta) have been implicated in the pathogenesis of Alzheimer's disease (AD). Although the conformation adopted by Abeta within these aggregates is not known, a beta-hairpin conformation is known to be accessible to monomeric Abeta. Here we show that this beta-hairpin is a building block of toxic Abeta oligomers by engineering a double-cysteine mutant (called Abetacc) in which the beta-hairpin is stabilized by an intramolecular disulfide bond. Abeta(40)cc and Abeta(42)cc both spontaneously form stable oligomeric species with distinct molecular weights and secondary-structure content, but both are unable to convert into amyloid fibrils. Biochemical and biophysical experiments and assays with conformation-specific antibodies used to detect Abeta aggregates in vivo indicate that the wild-type oligomer structure is preserved and stabilized in Abetacc oligomers. Stable oligomers are expected to become highly toxic and, accordingly, we find that beta-sheet-containing Abeta(42)cc oligomers or protofibrillar species formed by these oligomers are 50 times more potent inducers of neuronal apoptosis than amyloid fibrils or samples of monomeric wild-type Abeta(42), in which toxic aggregates are only transiently formed. The possibility of obtaining completely stable and physiologically relevant neurotoxic Abeta oligomer preparations will facilitate studies of their structure and role in the pathogenesis of AD. For example, here we show how kinetic partitioning into different aggregation pathways can explain why Abeta(42) is more toxic than the shorter Abeta(40), and why certain inherited mutations are linked to protofibril formation and early-onset AD.