Food protein-derived amyloids do not accelerate amyloid ß aggregation.

The deposition of proteins in the form of amyloid fibrils is closely associated with several serious diseases. The events that trigger the conversion from soluble functional proteins into insoluble amyloid are not fully understood. Many proteins that are not associated with disease can form amyloid with similar structural characteristics as the disease-associated fibrils, which highlights the potential risk of cross-seeding of disease amyloid by amyloid-like structures encountered in our surrounding. Of particular interest are common food proteins that can be transformed into amyloid under conditions similar to cooking. We here investigate cross-seeding of amyloid-ß (Aß), a peptide known to form amyloid during the development of Alzheimer's disease, by 16 types of amyloid fibrils derived from food proteins or peptides. Kinetic studies using thioflavin T fluorescence as output show that none of the investigated protein fibrils accelerates the aggregation of Aß. In at least two cases (hen egg lysozyme and oat protein isolate) we observe retardation of the aggregation, which appears to originate from interactions between the food protein seeds and Aß in aggregated form. The results support the view that food-derived amyloid is not a risk factor for development of Aß pathology and Alzheimer's disease.

Extracellular protein components of amyloid plaques and their roles in Alzheimer's disease pathology.

Alzheimer's disease (AD) is pathologically defined by the presence of fibrillar amyloid ß (Aß) peptide in extracellular senile plaques and tau filaments in intracellular neurofibrillary tangles. Extensive research has focused on understanding the assembly mechanisms and neurotoxic effects of Aß during the last decades but still we only have a brief understanding of the disease associated biological processes. This review highlights the many other constituents that, beside Aß, are accumulated in the plaques, with the focus on extracellular proteins. All living organisms rely on a delicate network of protein functionality. Deposition of significant amounts of certain proteins in insoluble inclusions will unquestionably lead to disturbances in the network, which may contribute to AD and copathology. This paper provide a comprehensive overview of extracellular proteins that have been shown to interact with Aß and a discussion of their potential roles in AD pathology. Methods that can expand the knowledge about how the proteins are incorporated in plaques are described. Top-down methods to analyze post-mortem tissue and bottom-up approaches with the potential to provide molecular insights on the organization of plaque-like particles are compared. Finally, a network analysis of Aß-interacting partners with enriched functional and structural key words is presented.

Enhanced detection of ATTR amyloid using a nanofibril-based assay.

More than 30 proteins and peptides have been found to form amyloid fibrils in human diseases. Fibrils formed by transthyretin (TTR) are associated with ATTR amyloidosis, affecting many vital organs, including the heart and peripheral nervous system. Congo red staining is the gold standard method for detection of amyloid deposits in tissue. However, Congo red staining and amyloid typing methods such as immunofluorescence labelling are limited to relatively large deposits. Detection of small ATTR deposits present at an early stage of the disease could enable timely treatment and prevent severe tissue damage. In this study, we developed an enhanced ATTR amyloid detection method that uses functionalised protein nanofibrils. Using this method, we achieved sensitive detection of monomeric TTR in a microplate immunoassay and immunofluorescence labelling of ex vivo tissue from two patients containing ATTR aggregates. The system's utility was confirmed on sections from a patient with AA amyloidosis and liver sections from inflamed mouse. These results suggest that the detection system constitutes important new technology for highly sensitive detection of microscopic amounts of ATTR amyloid deposited in tissue.

Protofibrillar and Fibrillar Amyloid-ß Binding Proteins in Cerebrospinal Fluid.

Aggregation and deposition of misfolded amyloid-ß (Aß) peptide in the brain is central to Alzheimer's disease (AD). Oligomeric, protofibrillar, and fibrillar forms of Aß are believed to be neurotoxic and cause neurodegeneration in AD, but the toxicity mechanisms are not well understood and may involve Aß-interacting molecular partners. In a previous study, we identified potential Aß42 protofibrillar-binding proteins in serum and cerebrospinal fluid (CSF) using an engineered version of Aß42 (Aß42CC) that forms protofibrils, but not fibrils. Here we studied binding of proteins to Aß42 fibrils in AD and non-AD CSF and compared these with protofibrillar Aß42CC-binding partners. Aß42 fibrils sequestered 2.4-fold more proteins than Aß42CC protofibrils. Proteins with selective binding to fibrillar aggregates with low nanomolar affinity were identified. We also found that protofibrillar and fibrillar Aß-binding proteins represent distinct functional categories. Aß42CC protofibrils triggered interactions with proteins involved in catalytic activities, like transferases and oxidoreductases, while Aß42 fibrils were more likely involved in binding to proteoglycans, growth factors and neuron-associated proteins, e.g., neurexin-1, -2, and -3. Interestingly, 10 brain-enriched proteins were identified among the fibril-binding proteins, while protofibril-extracted proteins had more general expression patterns. Both types of Aß aggregates bound several extracellular proteins. Additionally, we list a set of CSF proteins that might have potential to discriminate between AD and non-AD CSF samples. The results may be of relevance both for biomarker studies and for studies of Aß-related toxicity mechanisms.

Identification of proteins that specifically recognize and bind protofibrillar aggregates of amyloid-ß.

Protofibrils of the 42 amino acids long amyloid-ß peptide are transient pre-fibrillar intermediates in the process of peptide aggregation into amyloid plaques and are thought to play a critical role in the pathology of Alzheimer's disease. Hence, there is a need for research reagents and potential diagnostic reagents for detection and imaging of such aggregates. Here we describe an in vitro selection of Affibody molecules that bind to protofibrils of Aß42cc, which is a stable engineered mimic of wild type Aß42 protofibrils. Several binders were identified that bind Aß42cc protofibrils with low nanomolar affinities, and which also recognize wild type Aß42 protofibrils. Dimeric head-to-tail fusion proteins with subnanomolar binding affinities, and very slow dissociation off-rates, were also constructed. A mapping of the chemical properties of the side chains onto the Affibody scaffold surface reveals three distinct adjacent surface areas of positively charged surface, nonpolar surface and a polar surface, which presumably match a corresponding surface epitope on the protofibrils. The results demonstrate that the engineered Aß42cc is a suitable antigen for directed evolution of affinity reagents with specificity for wild type Aß42 protofibrils.

Binding of human proteins to amyloid-ß protofibrils.

The progressive neurodegeneration in Alzheimer's disease is believed to be linked to the presence of prefibrillar aggregates of the amyloid-ß (Aß) peptide in the brain. The exact role of these aggregates in the disease pathology is, however, still an open question. Any mechanism by which oligomeric Aß may cause damage to neuronal cells must, in one way or another, involve interactions with other molecules. Here, we identify proteins in human serum and cerebrospinal fluid that bind to stable protofibrils formed by an engineered variant of Aß42 (Aß42CC). We find that the protofibrils attract a substantial number of protein binding partners. Many of the 101 identified proteins are involved in lipid transport and metabolism, the complement system, or in hemostasis. Binding of representative proteins from all of these groups with micromolar affinity was confirmed using surface plasmon resonance. In addition, binding of apolipoprotein E to the protofibrils with nanomolar affinity was demonstrated. We also find that aggregation of Aß enhances protein binding, as lower amounts of proteins bind monomeric Aß. Proteins that bind to Aß protofibrils might contribute to biological effects in which these aggregates are involved. Our results therefore suggest that an improved understanding of the mechanisms by which Aß causes cytotoxicity and neurodegeneration might be gained from studies carried out in biologically relevant matrices in which Aß-binding proteins are present.

Amyloid-ß protofibrils: size, morphology and synaptotoxicity of an engineered mimic.

Structural and biochemical studies of the aggregation of the amyloid-ß peptide (Aß) 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 Aß called Aßcc, which forms stable protofibrils that do not convert into amyloid fibrils. Here we provide a detailed characterization of Aß42cc protofibrils. Like wild type Aß 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. Aß42cc 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 Aß42cc protofibrils into the same class of species as fibrillar oligomers of wild type Aß. Aß42cc protofibrils may be used to extract binding proteins in biological fluids and apolipoprotein E is readily detected as a binder in human serum. Finally, Aß42cc protofibrils act to attenuate spontaneous synaptic activity in mouse hippocampal neurons. The experiments indicate considerable structural and chemical similarities between protofibrils formed by Aß42cc and aggregates of wild type Aß42. We suggest that Aß42cc protofibrils may be used in research and applications that require stable preparations of protofibrillar Aß.