Sigurdson, C.J. et al. Prion strain discrimination using luminescent conjugated polymers. Nat. Meth. 4, 1023-1030

Department of Chemistry, Laval University, Quebec City, Quebec, Canada
Nature Methods (Impact Factor: 32.07). 01/2008; 4(12):1023-30. DOI: 10.1038/nmeth1131
Source: PubMed


The occurrence of multiple strains of prions may reflect conformational variability of PrP(Sc), a disease-associated, aggregated variant of the cellular prion protein, PrP(C). Here we used luminescent conjugated polymers (LCPs), which emit conformation-dependent fluorescence spectra, for characterizing prion strains. LCP reactivity and emission spectra of brain sections discriminated among four immunohistochemically indistinguishable, serially mouse-passaged prion strains derived from sheep scrapie, chronic wasting disease (CWD), bovine spongiform encephalopathy (BSE), and mouse-adapted Rocky Mountain Laboratory scrapie prions. Furthermore, using LCPs we differentiated between field isolates of BSE and bovine amyloidotic spongiform encephalopathy, and identified noncongophilic deposits in prion-infected deer and sheep. We found that fibrils with distinct morphologies generated from chemically identical recombinant PrP yielded unique LCP spectra, suggesting that spectral characteristic differences resulted from distinct supramolecular PrP structures. LCPs may help to detect structural differences among discrete protein aggregates and to link protein conformational features with disease phenotypes.

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    • "differences, as well as different disease phenotype and pathogenesis (Cobb and Surewicz 2009; Sigurdson et al. 2007). In a given host, both PrP C and PrP Sc are composed of identical amino acid sequences, so strain properties are maintained through conformational differences in PrP Sc (Bessen and Marsh 1994; Legname et al. 2005). "
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    ABSTRACT: Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal protein-misfolding neurodegenerative diseases. TSEs have been described in several species, including bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats, chronicwasting disease (CWD) in cervids, transmissible mink encephalopathy (TME) in mink, and Kuru and Creutzfeldt-Jakob disease (CJD) in humans. These diseases are associated with the accumulation of a protease-resistant, disease-associated isoform of the prion protein (called PrPSc) in the central nervous system and other tissues, depending on the host pecies. Typically, TSEs are acquired through exposure to infectious material, but inherited and spontaneous TSEs also occur. All TSEs share pathologic features and infectious mechanisms but have distinct differences in transmission and epidemiology due to host factors and strain differences encoded within the structure of the misfolded prion protein. The possibility that BSE can be transmitted to humans as the cause of variant Creutzfeldt-Jakob disease has brought attention to this family of diseases. This review is focused on the TSEs of livestock: bovine spongiform encephalopathy in cattle and scrapie in sheep and goats.
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    • "Hence, p-FTAA identified a wider range of p62-positive protein inclusion bodies compared to the conventional amyloid ligands Congo Red and ThS. These findings are in agreement with previous studies that have shown that thiophene-based amyloid ligands detect a larger subset of extracellular protein aggregates in tissue sections than Congo Red and derivatives of thioflavin.10, 36–39 An earlier report40 also demonstrated a difference in the staining pattern obtained with ThS and an anti-Aβ antibody in s-IBM muscle tissue. "
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    ABSTRACT: Small hydrophobic ligands identifying intracellular protein deposits are of great interest, as protein inclusion bodies are the pathological hallmark of several degenerative diseases. Here we report that fluorescent amyloid ligands, termed luminescent conjugated oligothiophenes (LCOs), rapidly and with high sensitivity detect protein inclusion bodies in skeletal muscle tissue from patients with sporadic inclusion body myositis (s-IBM). LCOs having a conjugated backbone of at least five thiophene units emitted strong fluorescence upon binding, and showed co-localization with proteins reported to accumulate in s-IBM protein inclusion bodies. Compared with conventional amyloid ligands, LCOs identified a larger fraction of immunopositive inclusion bodies. When the conjugated thiophene backbone was extended with terminal carboxyl groups, the LCO revealed striking spectral differences between distinct protein inclusion bodies. We conclude that 1) LCOs are sensitive, rapid and powerful tools for identifying protein inclusion bodies and 2) LCOs identify a wider range of protein inclusion bodies than conventional amyloid ligands.
    Full-text · Article · Mar 2013 · ChemBioChem
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    • "This methodology only needs a small fraction of labeled protein, thus, minimally altering the behavior of the native, unlabeled protein. This knowledge on the early aggregates could be completed for the late deposits by using fluorescence microscopy techniques, especially taking advantage of substances that specifically bind amyloidogenic material and change their fluorescent properties upon binding, such as de novo designed luminescent conjugated polymers [41,42]. Other microscopy techniques, such as fluorescence anisotropy microscopy [43] and fluorescence lifetime imaging [44], can report valuable data on the mature aggregates. "
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    ABSTRACT: Amyloidogenic protein aggregation is a persistent biomedical problem. Despite active research in disease-related aggregation, the need for multidisciplinary approaches to the problem is evident. Recent advances in single-molecule fluorescence spectroscopy are valuable for examining heterogenic biomolecular systems. In this work, we have explored the initial stages of amyloidogenic aggregation by employing fluorescence lifetime correlation spectroscopy (FLCS), an advanced modification of conventional fluorescence correlation spectroscopy (FCS) that utilizes time-resolved information. FLCS provides size distributions and kinetics for the oligomer growth of the SH3 domain of α-spectrin, whose N47A mutant forms amyloid fibrils at pH 3.2 and 37 °C in the presence of salt. The combination of FCS with additional fluorescence lifetime information provides an exciting approach to focus on the initial aggregation stages, allowing a better understanding of the fibrillization process, by providing multidimensional information, valuable in combination with other conventional methodologies.
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