A novel crosslinking protocol stabilizes amyloid ß oligomers capable of inducing Alzheimer's-associated pathologies.

Amyloid ß oligomers (AßOs) accumulate early in Alzheimer's disease (AD) and experimentally cause memory dysfunction and the major pathologies associated with AD, for example, tau abnormalities, synapse loss, oxidative damage, and cognitive dysfunction. In order to develop the most effective AßO-targeting diagnostics and therapeutics, the AßO structures contributing to AD-associated toxicity must be elucidated. Here, we investigate the structural properties and pathogenic relevance of AßOs stabilized by the bifunctional crosslinker 1,5-difluoro-2,4-dinitrobenzene (DFDNB). We find that DFDNB stabilizes synthetic Aß in a soluble oligomeric conformation. With DFDNB, solutions of Aß that would otherwise convert to large aggregates instead yield solutions of stable AßOs, predominantly in the 50-300 kDa range, that are maintained for at least 12 days at 37°C. Structures were determined by biochemical and native top-down mass spectrometry analyses. Assayed in neuronal cultures and i.c.v.-injected mice, the DFDNB-stabilized AßOs were found to induce tau hyperphosphorylation, inhibit choline acetyltransferase, and provoke neuroinflammation. Most interestingly, DFDNB crosslinking was found to stabilize an AßO conformation particularly potent in inducing memory dysfunction in mice. Taken together, these data support the utility of DFDNB crosslinking as a tool for stabilizing pathogenic AßOs in structure-function studies.

A human scFv antibody that targets and neutralizes high molecular weight pathogenic amyloid-ß oligomers.

Brain accumulation of soluble oligomers of the amyloid-ß peptide (AßOs) is increasingly considered a key early event in the pathogenesis of Alzheimer's disease (AD). A variety of AßO species have been identified, both in vitro and in vivo, ranging from dimers to 24mers and higher order oligomers. However, there is no consensus in the literature regarding which AßO species are most germane to AD pathogenesis. Antibodies capable of specifically recognizing defined subpopulations of AßOs would be a valuable asset in the identification, isolation, and characterization of AD-relevant AßO species. Here, we report the characterization of a human single chain antibody fragment (scFv) denoted NUsc1, one of a number of scFvs we have identified that stringently distinguish AßOs from both monomeric and fibrillar Aß. NUsc1 readily detected AßOs previously bound to dendrites in cultured hippocampal neurons. In addition, NUsc1 blocked AßO binding and reduced AßO-induced neuronal oxidative stress and tau hyperphosphorylation in cultured neurons. NUsc1 further distinguished brain extracts from AD-transgenic mice from wild type (WT) mice, and detected endogenous AßOs in fixed AD brain tissue and AD brain extracts. Biochemical analyses indicated that NUsc1 targets a subpopulation of AßOs with apparent molecular mass greater than 50 kDa. Results indicate that NUsc1 targets a particular AßO species relevant to AD pathogenesis, and suggest that NUsc1 may constitute an effective tool for AD diagnostics and therapeutics.