The Aß protofibril selective antibody mAb158 prevents accumulation of Aß in astrocytes and rescues neurons from Aß-induced cell death.

BACKGROUND: Currently, several amyloid beta (Aß) antibodies, including the protofibril selective antibody BAN2401, are in clinical trials. The murine version of BAN2401, mAb158, has previously been shown to lower the levels of pathogenic Aß and prevent Aß deposition in animal models of Alzheimer's disease (AD). However, the cellular mechanisms of the antibody's action remain unknown. We have recently shown that astrocytes effectively engulf Aß42 protofibrils, but store rather than degrade the ingested Aß aggregates. In a co-culture set-up, the incomplete degradation of Aß42 protofibrils by astrocytes results in increased neuronal cell death, due to the release of extracellular vesicles, containing N-truncated, neurotoxic Aß. METHODS: The aim of the present study was to investigate if the accumulation of Aß in astrocytes can be affected by the Aß protofibril selective antibody mAb158. Co-cultures of astrocytes, neurons, and oligodendrocytes, derived from embryonic mouse cortex, were exposed to Aß42 protofibrils in the presence or absence of mAb158. RESULTS: Our results demonstrate that the presence of mAb158 almost abolished Aß accumulation in astrocytes. Consequently, mAb158 treatment rescued neurons from Aß-induced cell death. CONCLUSION: Based on these findings, we conclude that astrocytes may play a central mechanistic role in anti-Aß immunotherapy.

Stabilization of neurotoxic Alzheimer amyloid-beta oligomers by protein engineering.

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.

Accumulation of amyloid-ß by astrocytes result in enlarged endosomes and microvesicle-induced apoptosis of neurons.

BACKGROUND: Despite the clear physical association between activated astrocytes and amyloid-ß (Aß) plaques, the importance of astrocytes and their therapeutic potential in Alzheimer's disease remain elusive. Soluble Aß 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 Aß. RESULTS: To examine the role of astrocytes in Aß pathology, we added Aß protofibrils to a co-culture system of primary neurons and glia. Our data demonstrates that astrocytes rapidly engulf large amounts of Aß 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 Aß and severe lysosomal dysfunction. Moreover, secretion of microvesicles containing N-terminally truncated Aß, 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 Aß species.

Increased Number of Plasma B Cells Producing Autoantibodies Against Aß42 Protofibrils in Alzheimer's Disease.

The Alzheimer's disease (AD)-related peptide amyloid-ß (Aß) has a propensity to aggregate into various assemblies including toxic soluble Aß protofibrils. Several studies have reported the existence of anti-Aß antibodies in humans. However, it is still debated whether levels of anti-Aß antibodies are altered in AD patients compared to healthy individuals. Formation of immune complexes with plasma Aß makes it difficult to reliably measure the concentration of circulating anti-Aß antibodies with certain immunoassays, potentially leading to an underestimation. Here we have investigated anti-Aß antibody production on a cellular level by measuring the amount of anti-Aß antibody producing cells instead of the plasma level of anti-Aß antibodies. To our knowledge, this is the first time the anti-Aß 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 Aß40 monomers, whereas the number of cells producing antibodies toward Aß42 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 Aß protofibrils, which is significantly increased in AD patients.

Interference from heterophilic antibodies in amyloid-ß oligomer ELISAs.

Amyloid-ß (Aß) oligomers of different sizes and forms have recently been the focus formany Alzheimer's disease (AD) researchers. Various immunoassays have been used to detect low concentrations of these elusive Aß 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 Aß 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.

Diverse foxp3 expression in children with type 1 diabetes and celiac disease.

Imbalance between different types of T lymphocytes, such as T helper (Th) and regulatory T cells (Tregs), has been reported to play a part in the pathogenesis behind such autoimmune diseases as type 1 diabetes (T1D) and celiac disease (CD). Defects in Tregs are proposed to at least partly explain the imbalance of Th cells found in children with immunologic diseases. Peripheral blood mononuclear cells from 24 children with T1D and/or CD, and reference children (that is, those without any of these diseases) were stimulated with disease-associated antigens (insulin, gluten, transglutaminase [tTG]), and phytohemagglutinin (PHA). The mRNA expression of the Treg-associated marker FOXP3 was analyzed with multiplex real-time RT-PCR. Children with T1D showed both a low spontaneous (P < 0.05) and PHA-induced (P < 0.01) expression of FOXP3 mRNA compared to children with CD. Children with T1D also had a low PHA-induced FOXP3 mRNA expression compared to the group of children diagnosed with both T1D and CD (P < 0.05). Spontaneous (P < 0.05) and PHA-induced (P < 0.05) FOXP3 mRNA expression was high in children with CD compared to reference children. In contrast, stimulation with insulin tended to induce high FOXP3 mRNA expression in T1D children compared to reference children (P= 0.057). In conclusion, children with only T1D generally showed a lower FOXP3 mRNA expression than did children with CD, or with T1D in combination with CD, which suggests impaired regulation of the immune system in children with T1D.