A generic method for design of oligomer-specific antibodies.

Antibodies that preferentially and specifically target pathological oligomeric protein and peptide assemblies, as opposed to their monomeric and amyloid counterparts, provide therapeutic and diagnostic opportunities for protein misfolding diseases. Unfortunately, the molecular properties associated with oligomer-specific antibodies are not well understood, and this limits targeted design and development. We present here a generic method that enables the design and optimisation of oligomer-specific antibodies. The method takes a two-step approach where discrimination between oligomers and fibrils is first accomplished through identification of cryptic epitopes exclusively buried within the structure of the fibrillar form. The second step discriminates between monomers and oligomers based on differences in avidity. We show here that a simple divalent mode of interaction, as within e.g. the IgG isotype, can increase the binding strength of the antibody up to 1500 times compared to its monovalent counterpart. We expose how the ability to bind oligomers is affected by the monovalent affinity and the turnover rate of the binding and, importantly, also how oligomer specificity is only valid within a specific concentration range. We provide an example of the method by creating and characterising a spectrum of different monoclonal antibodies against both the Aß peptide and α-synuclein that are associated with Alzheimer's and Parkinson's diseases, respectively. The approach is however generic, does not require identification of oligomer-specific architectures, and is, in essence, applicable to all polypeptides that form oligomeric and fibrillar assemblies.

Ca(2+) enhances Aß polymerization rate and fibrillar stability in a dynamic manner.

Identifying factors that affect the self-assembly of Aß (amyloid-ß peptide) is of utmost importance in the quest to understand the molecular mechanisms causing AD (Alzheimer's disease). Ca(2+) has previously been shown to accelerate both Aß fibril nucleation and maturation, and dysregulated Ca(2+) homoeostasis frequently correlates with development of AD. The mechanisms regarding Ca(2+) binding, as well as its effect on fibril kinetics, are not fully understood. Using a polymerization assay we show that Ca(2+) in a dynamic and reversible manner enhances both the elongation rate and fibrillar stability, where specifically the 'dock and lock' phase mechanism is enhanced. Through NMR analysis we found that Ca(2+) affects the fibrillar architecture. In addition, and unexpectedly, we found that Ca(2+) does not bind the free Aß monomer. This implies that Ca(2+) binding requires an architecture adopted by assembled peptides, and consequently is mediated through intermolecular interactions between adjacent peptides. This gives a mechanistic explanation to the enhancing effect on fibril maturation and indicates structural similarities between prefibrillar structures and mature amyloid. Taken together we show how Ca(2+) levels affect the delicate equilibrium between the monomeric and assembled Aß and how fluctuations in vivo may contribute to development and progression of the disease.

Functional links between Aß toxicity, endocytic trafficking, and Alzheimer's disease risk factors in yeast.

Aß (beta-amyloid peptide) is an important contributor to Alzheimer's disease (AD). We modeled Aß toxicity in yeast by directing the peptide to the secretory pathway. A genome-wide screen for toxicity modifiers identified the yeast homolog of phosphatidylinositol binding clathrin assembly protein (PICALM) and other endocytic factors connected to AD whose relationship to Aß was previously unknown. The factors identified in yeast modified Aß toxicity in glutamatergic neurons of Caenorhabditis elegans and in primary rat cortical neurons. In yeast, Aß impaired the endocytic trafficking of a plasma membrane receptor, which was ameliorated by endocytic pathway factors identified in the yeast screen. Thus, links between Aß, endocytosis, and human AD risk factors can be ascertained with yeast as a model system.

Amyloid-ß oligomer specificity mediated by the IgM isotype--implications for a specific protective mechanism exerted by endogenous auto-antibodies.

BACKGROUND: Alzheimers disease (AD) has been strongly linked to an anomalous self-assembly of the amyloid-ß peptide (Aß). The correlation between clinical symptoms of AD and Aß depositions is, however, weak. Instead small and soluble Aß oligomers are suggested to exert the major pathological effects. In strong support of this notion, immunological targeting of Aß oligomers in AD mice-models shows that memory impairments can be restored without affecting the total burden of Aß deposits. Consequently a specific immunological targeting of Aß oligomers is of high therapeutic interest. METHODOLOGY/PRINCIPAL FINDINGS: Previously the generation of conformational-dependent oligomer specific anti-Aß antibodies has been described. However, to avoid the difficult task of identifying a molecular architecture only present on oligomers, we have focused on a more general approach based on the hypothesis that all oligomers expose multiple identical epitopes and therefore would have an increased binding to a multivalent receptor. Using the polyvalent IgM immunoglobulin we have developed a monoclonal anti-Aß antibody (OMAB). OMAB only demonstrates a weak interaction with Aß monomers and dimers having fast on and off-rate kinetics. However, as an effect of avidity, its interaction with Aß-oligomers results in a strong complex with an exceptionally slow off-rate. Through this mechanism a selectivity towards Aß oligomers is acquired and OMAB fully inhibits the cytotoxic effect exerted by Aß(1-42) at highly substoichiometric ratios. Anti-Aß auto-antibodies of IgM isotype are frequently present in the sera of humans. Through a screen of endogenous anti-Aß IgM auto-antibodies from a group of healthy individuals we show that all displays a preference for oligomeric Aß. CONCLUSIONS/SIGNIFICANCE: Taken together we provide a simple and general mechanism for targeting of oligomers without the requirement of conformational-dependent epitopes. In addition, our results suggest that IgM anti-Aß auto-antibodies may exert a more specific protective mechanism in vivo than previously anticipated.

Quenched hydrogen/deuterium exchange NMR characterization of amyloid-beta peptide aggregates formed in the presence of Cu2+ or Zn2+.

Alzheimer's disease, a neurodegenerative disorder causing synaptic impairment and neuronal cell death, is strongly correlated with aggregation of the amyloid-beta peptide (Abeta). Divalent metal ions such as Cu(2+) and Zn(2+) are known to significantly affect the rate of aggregation and morphology of Abeta assemblies in vitro and are also found at elevated levels within cerebral plaques in vivo. The present investigation characterized the architecture of the aggregated forms of Abeta(1-40) and Abeta(1-42) in the presence or absence of either Cu(2+) or Zn(2+) using quenched hydrogen/deuterium exchange combined with solution NMR spectroscopy. The NMR analyses provide a quantitative and residue-specific structural characterization of metal-induced Abeta aggregates, showing that both the peptide sequence and the type of metal ion exert an impact on the final architecture. Common features among the metal-complexed peptide aggregates are two solvent-protected regions with an intervening minimum centered at Asn27, and a solvent-accessible N-terminal region, Asp1-Lys16. Our results suggest that Abeta in complex with either Cu(2+) or Zn(2+) can attain an aggregation-prone beta-strand-turn-beta-strand motif, similar to the motif found in fibrils, but where the metal binding to the N-terminal region guides the peptide into an assembly distinctly different from the fibril form.

Amide solvent protection analysis demonstrates that amyloid-beta(1-40) and amyloid-beta(1-42) form different fibrillar structures under identical conditions.

AD (Alzheimer's disease) is a neurodegenerative disorder characterized by self-assembly and amyloid formation of the 39-43 residue long Abeta (amyloid-beta)-peptide. The most abundant species, Abeta(1-40) and Abeta(1-42), are both present within senile plaques, but Abeta(1-42) peptides are considerably more prone to self-aggregation and are also essential for the development of AD. To understand the molecular and pathological mechanisms behind AD, a detailed knowledge of the amyloid structures of Abeta-peptides is vital. In the present study we have used quenched hydrogen/deuterium-exchange NMR experiments to probe the structure of Abeta(1-40) fibrils. The fibrils were prepared and analysed identically as in our previous study on Abeta(1-42) fibrils, allowing a direct comparison of the two fibrillar structures. The solvent protection pattern of Abeta(1-40) fibrils revealed two well-protected regions, consistent with a structural arrangement of two beta-strands connected with a bend. This protection pattern partly resembles the pattern found in Abeta(1-42) fibrils, but the Abeta(1-40) fibrils display a significantly increased protection for the N-terminal residues Phe4-His14, suggesting that additional secondary structure is formed in this region. In contrast, the C-terminal residues Gly37-Val40 show a reduced protection that suggests a loss of secondary structure in this region and an altered filament assembly. The differences between the present study and other similar investigations suggest that subtle variations in fibril-preparation conditions may significantly affect the fibrillar architecture.