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 Aβ1-42 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 (Aβ 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.

The molecular architecture of amyloids formed in vivo can be interrogated using luminescent conjugated oligothiophenes (LCOs), a unique class of amyloid dyes. When bound to amyloid, LCOs yield fluorescence emission spectra that reflect the 3D structure of the protein aggregates. Given that synthetic amyloid-β peptide (Aβ) has been shown to adopt distinct structural conformations with different biological activities, we asked whether Aβ can assume structurally and functionally distinct conformations within the brain. To this end, we analyzed the LCO-stained cores of β-amyloid plaques in postmortem tissue sections from frontal, temporal, and occipital neocortices in 40 cases of familial Alzheimer's disease (AD) or sporadic (idiopathic) AD (sAD). The spectral attributes of LCO-bound plaques varied markedly in the brain, but the mean spectral properties of the amyloid cores were generally similar in all three cortical regions of individual patients. Remarkably, the LCO amyloid spectra differed significantly among some of the familial and sAD subtypes, and between typical patients with sAD and those with posterior cortical atrophy AD. Neither the amount of Aβ nor its protease resistance correlated with LCO spectral properties. LCO spectral amyloid phenotypes could be partially conveyed to Aβ plaques induced by experimental transmission in a mouse model. These findings indicate that polymorphic Aβ-amyloid deposits within the brain cluster as clouds of conformational variants in different AD cases. Heterogeneity in the molecular architecture of pathogenic Aβ among individuals and in etiologically distinct subtypes of AD justifies further studies to assess putative links between Aβ conformation and clinical phenotype.

Deposition of aggregated Aβ 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 Aβ 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.

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Using luminescent conjugated polyelectrolyte probes (LCPs), we demonstrate the possibility to distinguish amyloid-beta 1-42 peptide (Abeta1-42) fibril conformations, by analyzing in vitro generated amyloid fibrils of Abeta1-42 formed under quiescent and agitated conditions. LCPs were then shown to resolve such conformational heterogeneity of amyloid deposits in vivo. A diversity of amyloid deposits depending upon morphology and anatomic location was illustrated with LCPs in frozen ex vivo brain sections from a transgenic mouse model (tg-APP swe) of Alzheimer's disease. Comparative LCP fluorescence showed that compact-core plaques of amyloid beta precursor protein transgenic mice were composed of rigid dense amyloid. A more abundant form of amyloid plaque displayed morphology of a compact center with a protruding diffuse exterior. Surprisingly, the compact center of these plaques showed disordered conformations of the fibrils, and the exterior was composed of rigid amyloid protruding from the disordered center. This type of plaque appears to grow from more loosely assembled regions toward solidified amyloid tentacles. This work demonstrates how application of LCPs can prove helpful to monitor aggregate structure of in vivo formed amyloid deposits such as architecture, maturity, and origin.