Nanoscale Structure and Spectroscopic Probing of Aß1-40 Fibril Bundle Formation.

Amyloid plaques composed of fibrillar Amyloid-ß (Aß) are hallmarks of Alzheimer's disease. However, Aß fibrils are morphologically heterogeneous. Conformation sensitive luminescent conjugated oligothiophenes (LCOs) are versatile tools for monitoring such fibril polymorphism in vivo and in vitro. Biophysical methods applied on in vitro generated Aß fibrils, stained with LCOs with different binding and fluorescence properties, can be used to characterize the Aß fibrillation in depth, far beyond that possible for in vivo generated amyloid plaques. In this study, in vitro fibrillation of the Aß1-40 peptide was monitored by time-lapse transmission electron microscopy, LCO fluorescence, and atomic force microscopy. Differences in the LCO binding in combination with nanoscale imaging revealed that spectral variation correlated with fibrils transforming from solitary filaments (Ø~2.5 nm) into higher order bundled structures (Ø~5 nm). These detailed in vitro experiments can be used to derive data that reflects the heterogeneity of in vivo generated Aß plaques observed by LCO fluorescence. Our work provides new structural basis for targeted drug design and molecular probe development for amyloid imaging.

(11)C and (18)F Radiolabeling of Tetra- and Pentathiophenes as PET-Ligands for Amyloid Protein Aggregates.

Three oligothiophenes were evaluated as PET ligands for the study of local and systemic amyloidosis ex vivo using tissue from patients with amyloid deposits and in vivo using healthy animals and PET-CT. The ex vivo binding studies revealed that all three labeled compounds bound specifically to human amyloid deposits. Specific binding was found in the heart, kidney, liver, and spleen. To verify the specificity of the oligothiophenes toward amyloid deposits, tissue sections with amyloid pathology were stained using the fluorescence exhibited by the compounds and evaluated with multiphoton microscopy. Furthermore, a in vivo monkey PET-CT study showed very low uptake in the brain, pancreas, and heart of the healthy animal indicating low nonspecific binding to healthy tissue. The biological evaluations indicated that this is a promising group of compounds for the visualization of systemic and localized amyloidosis.

An azide functionalized oligothiophene ligand--a versatile tool for multimodal detection of disease associated protein aggregates.

Ligands for identifying protein aggregates are of great interest as such deposits are the pathological hallmark of a wide range of severe diseases including Alzheimer's and Parkinson's disease. Here we report the synthesis of an azide functionalized fluorescent pentameric oligothiophene that can be utilized as a ligand for multimodal detection of disease-associated protein aggregates. The azide functionalization allows for attachment of the ligand to a surface by conventional click chemistry without disturbing selective interaction with protein aggregates and the oligothiophene-aggregate interaction can be detected by fluorescence or surface plasmon resonance. In addition, a methodology where the oligothiophene ligand is employed as a capturing molecule selective for aggregated proteins in combination with an antibody detecting a distinct peptide/protein is also presented. We foresee that this methodology will offer the possibility to create a variety of multiplex sensing systems for sensitive and selective detection of protein aggregates, the pathological hallmarks of several neurodegenerative diseases.

Evidence for age-dependent in vivo conformational rearrangement within Aß amyloid deposits.

Deposition of aggregated Aß peptide in the brain is one of the major hallmarks of Alzheimer's disease. Using a combination of two structurally different, but related, hypersensitive fluorescent amyloid markers, LCOs, reporting on separate ultrastructural elements, we show that conformational rearrangement occurs within Aß plaques of transgenic mouse models as the animals age. This important mechanistic insight should aid the design and evaluation of experiments currently using plaque load as readout.

Synthesis of a library of oligothiophenes and their utilization as fluorescent ligands for spectral assignment of protein aggregates.

Molecular probes for selective identification of protein aggregates are important to advance our understanding of the molecular pathogenesis underlying protein aggregation diseases. Here we report the chemical design of a library of anionic luminescent conjugated oligothiophenes (LCOs), which can be utilized as ligands for detection of protein aggregates. Certain molecular requirements were shown to be necessary for detecting (i) early non-thioflavinophilic protein assemblies of Aß1-42 and insulin preceding the formation of amyloid fibrils and (ii) for obtaining distinct spectral signatures of the two main pathological hallmarks observed in human Alzheimer's diease brain tissue (Aß plaques and neurofibrillary tangles). Our findings suggest that a superior anionic LCO-based ligand should have a backbone consisting of five to seven thiophene units and carboxyl groups extending the conjugated thiophene backbone. Such LCOs will be highly useful for studying the underlying molecular events of protein aggregation diseases and could also be utilized for the development of novel diagnostic tools for these diseases.