Multimodal fluorescence microscopy of prion strain specific PrP deposits stained by thiophene-based amyloid ligands

Abstract

The disease-associated prion protein (PrP) forms aggregates which vary in structural conformation yet share an identical primary sequence. These variations in PrP conformation are believed to manifest in prion strains exhibiting distinctly different periods of disease incubation as well as regionally specific aggregate deposition within the brain. The anionic luminescent conjugated polythiophene (LCP), polythiophene acetic acid (PTAA) has previously been used to distinguish PrP deposits associated with distinct mouse adapted strains via distinct fluorescence emission profiles from the dye. Here, we employed PTAA and 3 structurally related chemically defined luminescent conjugated oligothiophenes (LCOs) to stain brain tissue sections from mice inoculated with 2 distinct prion strains. Our results showed that in addition to emission spectra, excitation, and fluorescence lifetime imaging microscopy (FLIM) can fruitfully be assessed for optical distinction of PrP deposits associated with distinct prion strains. Our findings support the theory that alterations in LCP/LCO fluorescence are due to distinct conformational restriction of the thiophene backbone upon interaction with PrP aggregates associated with distinct prion strains. We foresee that LCP and LCO staining in combination with multimodal fluorescence microscopy might aid in detecting structural differences among discrete protein aggregates and in linking protein conformational features with disease phenotypes for a variety of neurodegenerative proteinopathies.

Amyloid-β and lysozyme proteotoxicity in Drosophila : Beneficial effects of lysozyme and serum amyloid P component in models of Alzheimer’s disease and lysozyme amyloidosis

“…Interestingly, a shorter fluorescence decay was observed for the h-FTAA positive prion aggregates in the sheep scrapie mice compared with the fluorescence decay observed for aggregates detected in a mouse model for chronic wasting disease (CWD). The terminal onset for scrapie mice occurs much earlier than for CWD mice (Sigurdson et al, 2007), indicating that there is a greater toxic effect in the scrapie mice, in which h-FTAA fluorescence decay was observed to be shorter compared to that observed in the CWD mice (Magnusson et al, 2014). Since the shortest fluorescence decay was observed for the lysozyme species detected in the F57I-F57I flies, this strengthens our hypothesis that these species possess toxic properties.…”

“…LCOs are conjugated systems whose flexible thiophene backbone can adopt distinct conformations when binding to different structures, and these conformations can be observed as alterations in the optical properties, e.g. the emission spectra and the fluorescence lifetime, of the dye (Arja et al, 2013;Åslund et al, 2009;Magnusson et al, 2014). Two LCOs were used in the papers included in this thesis: p-and h-FTAA.…”

“…Fluorescence decay data for the different genotypes can be seen in table 6. FLIM analysis of two different mouse models for prion disease has been performed using h-FTAA, and h-FTAA was found to give rise to distinct fluorescence decay lifetimes depending on what prion strain the aggregates were associated with (Magnusson et al, 2014). Interestingly, a shorter fluorescence decay was observed for the h-FTAA positive prion aggregates in the sheep scrapie mice compared with the fluorescence decay observed for aggregates detected in a mouse model for chronic wasting disease (CWD).…”

Porcine prion protein amyloid

“…It is well established that amyloid fibrils show extensive structural polymorphism and is similarly believed to manifest as different pathogenic states representing prion strains in prion diseases. 11 The plethora of molecular structures of PrP in infectious and transmissible prions has recently been reviewed by Baskakov and co-workers. 8 A consensus structural feature from several studies of APrP is a cross-beta-sheet structure of the C-terminal sequence stretching from a variable starting point from the N-terminus (residue 90-140) all the way toward the final residue 231 within PrP fibrils.…”

The influence of lysozyme and oligothiophenes on amyloid-β toxicity in models of Alzheimer’s disease

“…This optical behavior can be used to discriminate between different protein aggregates. For instance, LCOs have been shown to spectrally discriminate between different prion strains [194] and the LCO p-FTAA can distinguish between Aβ deposits and NFT [195]. In addition, LCOs can be used to monitor the aggregation of different amyloid proteins.…”