Stabilization of neurotoxic Alzheimer amyloid- oligomers by protein engineering

Institute of Biomedicine, University of Gothenburg, SE-405 30 Gothenburg, Sweden.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 08/2010; 107(35):15595-600. DOI: 10.1073/pnas.1001740107
Source: PubMed


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.

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Available from: Anatoly Dubnovitsky, Oct 09, 2015
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    • "The arrow indicates the band corresponding to apolipoprotein E. Essentially no binding is observed in control experiments with glycine-coated beads (-PF). The strong bands around 15 kDa are SDS-stable dimers and trimers of Aβ42cc, as reported previously [16]. "
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    ABSTRACT: Structural and biochemical studies of the aggregation of the amyloid-β peptide (Aβ) are important to understand the mechanisms of Alzheimer's disease, but research is complicated by aggregate inhomogeneity and instability. We previously engineered a hairpin form of Aβ called Aβcc, which forms stable protofibrils that do not convert into amyloid fibrils. Here we provide a detailed characterization of Aβ42cc protofibrils. Like wild type Aβ they appear as smooth rod-like particles with a diameter of 3.1 (±0.2) nm and typical lengths in the range 60 to 220 nm when observed by atomic force microscopy. Non-perturbing analytical ultracentrifugation and nanoparticle tracking analyses are consistent with such rod-like protofibrils. Aβ42cc protofibrils bind the ANS dye indicating that they, like other toxic protein aggregates, expose hydrophobic surface. Assays with the OC/A11 pair of oligomer specific antibodies put Aβ42cc protofibrils into the same class of species as fibrillar oligomers of wild type Aβ. Aβ42cc protofibrils may be used to extract binding proteins in biological fluids and apolipoprotein E is readily detected as a binder in human serum. Finally, Aβ42cc protofibrils act to attenuate spontaneous synaptic activity in mouse hippocampal neurons. The experiments indicate considerable structural and chemical similarities between protofibrils formed by Aβ42cc and aggregates of wild type Aβ42. We suggest that Aβ42cc protofibrils may be used in research and applications that require stable preparations of protofibrillar Aβ.
    PLoS ONE 10/2013; 8(7):e66101. DOI:10.1371/journal.pone.0066101 · 3.23 Impact Factor
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    • "As additional proof that Aβ has biological effects that are sequence and conformation specific, in the presence of the inverse sequence Aβ35−25 we added into the perfusion system the regular sequence Aβ25−35 [1 µM] which, unsurprisingly, significantly reduced the power of the cholinergic theta oscillatory activity to approximately one-third of control cholinergic values. Finally, in relation to the differences in biological activity between Aβ25−35 and Aβ1−42, the latter always produced a reduction of theta oscillatory activity, and, furthermore, it was more effective at a lower concentration than Aβ25−35, which is consistent with the idea that the most toxic Aβ species is soluble Aβ1−42 [96, 97], especially in its oligomerized form [98, 99]. In this study, both peptides were used in their soluble forms, which have been shown to be mainly composed of monomers and a few oligomers [100], although the precise concentrations of each aggregation species are hard to determine. "
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    ABSTRACT: Soluble amyloid beta peptide (A β ) is responsible for the early cognitive dysfunction observed in Alzheimer's disease. Both cholinergically and glutamatergically induced hippocampal theta rhythms are related to learning and memory, spatial navigation, and spatial memory. However, these two types of theta rhythms are not identical; they are associated with different behaviors and can be differentially modulated by diverse experimental conditions. Therefore, in this study, we aimed to investigate whether or not application of soluble A β alters the two types of theta frequency oscillatory network activity generated in rat hippocampal slices by application of the cholinergic and glutamatergic agonists carbachol or DHPG, respectively. Due to previous evidence that oscillatory activity can be differentially affected by different A β peptides, we also compared Aβ 25-35 and Aβ 1-42 for their effects on theta rhythms in vitro at similar concentrations (0.5 to 1.0 μ M). We found that Aβ 25-35 reduces, with less potency than Aβ 1-42, carbachol-induced population theta oscillatory activity. In contrast, DHPG-induced oscillatory activity was not affected by a high concentration of Aβ 25-35 but was reduced by Aβ 1-42. Our results support the idea that different amyloid peptides might alter specific cellular mechanisms related to the generation of specific neuronal network activities, instead of exerting a generalized inhibitory effect on neuronal network function.
    International Journal of Peptides 06/2013; 2013(1):328140. DOI:10.1155/2013/328140
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    • "However, there is no conclusive measurement of the structure of the monomers and the oligomers under physiological conditions. Hard and coworkers have suggested that forcing the monomer into a hairpin-like structure increases its toxicity (Sandberg et al., 2010). Smith and coworkers have suggested that different oligomeric species of similar size can have dissimilar toxicity (Ladiwala et al., 2012), pointing toward the role played by folding. "
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    ABSTRACT: Amyloid beta (Aβ) is an extracellular 39-43 residue long peptide present in the mammalian cerebrospinal fluid, whose aggregation is associated with Alzheimer's disease (AD). Small oligomers of Aβ are currently thought to be the key to toxicity. However, it is not clear why the monomers of Aβ are non-toxic, and at what stage of aggregation toxicity emerges. Interactions of Aβ with cell membranes is thought to be the initiator of toxicity, but membrane binding studies with different preparations of monomers and oligomers have not settled this issue. We have earlier found that thermodynamically stable Aβ monomers emerge spontaneously from oligomeric mixtures upon long term incubation in physiological solutions (Nag et al., 2011). Here we show that the membrane-affinity of these stable Aβ monomers is much lower than that of a mixture of monomers and small oligomers (containing dimers to decamers), providing a clue to the emergence of toxicity. Fluorescently labeled Aβ40 monomers show negligible binding to cell membranes of a neuronal cell line (RN46A) at physiological concentrations (250 nM), while oligomers at the same concentrations show strong binding within 30 min of incubation. The increased affinity most likely does not require any specific neuronal receptor, since this difference in membrane-affinity was also observed in a somatic cell-line (HEK 293T). Similar results are also obtained for Aβ42 monomers and oligomers. Minimal amount of cell death is observed at these concentrations even after 36 h of incubation. It is likely that membrane binding precedes subsequent slower toxic events induced by Aβ. Our results (a) provide an explanation for the non-toxic nature of Aβ monomers, (b) suggest that Aβ toxicity emerges at the initial oligomeric phase, and (c) provide a quick assay for monitoring the benign-to-toxic transformation of Aβ.
    Frontiers in Physiology 04/2013; 4:84. DOI:10.3389/fphys.2013.00084 · 3.53 Impact Factor
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