The identification of disease-causing mutations in Alzheimers disease has contributed greatly to the understanding of the pathogenesis of this disease. The amyloid-? (A?) peptide has come into focus and is believed to be central to the pathogenesis of Alzheimers disease. With only symptomatic treatment available, efforts to develop new therapeutics aimed at lowering the amount of A? peptides in the affected brain have intensified. In particular, immunotherapy against A? peptides has attracted considerable interest, as it offers the possibility to generate highly specific molecules targeting highly specific moieties. Due to intense research efforts and massive investments at universities and in the pharmaceutical industry, the outlook for patients and their relatives has never been brighter.
Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of A? peptides are cornerstones in the pathology of Alzheimers disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of A? protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity.
We have investigated the use of isoelectric focusing and immunodetection for the separation of low molecular weight species of amyloid-beta (Abeta) peptides from their aggregates. From solutions of Abeta(1-40) or Abeta(1-42) monomeric peptides, low molecular weight material appeared at a pI value of ca. 5, while the presence of aggregates was detected as bands, observed at a pI of 6-6.5. The formation of Abeta aggregates (protofibrils) was verified by a sandwich ELISA, employing the protofibril conformation-selective antibody mAb158. In order to study the aggregation behavior when using a mixture of the monomers, we utilized the IEF separation combined with Western blot using two polyclonal antisera, selective for Abeta(1-40) and Abeta(1-42), respectively. We conclude that both monomers were incorporated in the aggregates. In a further study of the mixed aggregates, we used the protofibril conformation-selective antibody mAb158 for immunoprecipitation, followed by nanoelectrospray mass spectrometry (IP-MS). This showed that the Abeta(1-42) peptide is incorporated in the aggregate in a significantly larger proportion than its relative presence in the original monomer composition. IP-MS with mAb158 was also performed, and compared to IP-MS with the Abeta-selective antibody mAb1C3, where a monomeric Abeta(1-16) peptide was added to the protofibril preparation. Abeta(1-16) is known for its poor aggregation propensity, and acted therefore as a selectivity marker. The results obtained confirmed the protofibril conformation selectivity of mAb158.
Amyloid-? (A?) protofibrils are neurotoxic soluble intermediates in the A? aggregation process eventually forming senile plaques in Alzheimers disease. This A? species is a potential biomarker for Alzheimers disease and also a promising target for immunotherapy. In this study, we investigated the characteristics of conformation-dependent A? antibodies specific for A? protofibrils.
Human genetics link Alzheimers disease pathogenesis to excessive accumulation of amyloid-beta (Abeta) in brain, but the symptoms do not correlate with senile plaque burden. Since soluble Abeta aggregates can cause synaptic dysfunctions and memory deficits, these species could contribute to neuronal dysfunction and dementia. Here we explored selective targeting of large soluble aggregates, Abeta protofibrils, as a new immunotherapeutic strategy. The highly protofibril-selective monoclonal antibody mAb158 inhibited in vitro fibril formation and protected cells from Abeta protofibril-induced toxicity. When the mAb158 antibody was administered for 4 months to plaque-bearing transgenic mice with both the Arctic and Swedish mutations (tg-ArcSwe), Abeta protofibril levels were lowered while measures of insoluble Abeta were unaffected. In contrast, when treatment began before the appearance of senile plaques, amyloid deposition was prevented and Abeta protofibril levels diminished. Therapeutic intervention with mAb158 was however not proven functionally beneficial, since place learning depended neither on treatment nor transgenicity. Our findings suggest that Abeta protofibrils can be selectively cleared with immunotherapy in an animal model that display highly insoluble Abeta deposits, similar to those of Alzheimers disease brain.
The lowering of natively analyzed Abeta42 in cerebrospinal fluid (CSF) is used as a diagnostic tool in Alzheimers disease (AD). The presence of Abeta oligomers can interfere with such analyses causing underestimation of Abeta levels due to epitope masking. The aim was to investigate if the lowering of CSF Abeta42 seen in AD is caused by oligomerization.
Soluble amyloid-? (A?) aggregates of various sizes, ranging from dimers to large protofibrils, have been associated with neurotoxicity and synaptic dysfunction in Alzheimers Disease (AD). To investigate the properties of biologically relevant A? species, brain extracts from amyloid ? protein precursor (A?PP) transgenic mice and AD patients as well as synthetic A? 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 A?42 was divided in four fractions, with large aggregates in fraction 1 and the smallest species in fraction 4. Synthetic A? aggregates from fractions 2 and 3 proved to be most toxic in an MTT assay. In A?PP transgenic mouse brain, the most abundant soluble A? species were found in fraction 2 and consisted mainly of A?40. Also in AD brains, A? was mainly found in fraction 2 but primarily as A?42. All biologically derived A? from fraction 2 was immunologically discriminated from smaller species with mAb158. Thus, the predominant species of biologically derived soluble A?, natively separated by density gradient ultracentrifugation, were found to match the size of the neurotoxic, 80-500 kDa synthetic A? protofibrils and were equally detected with mAb158.
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