Sigurdson, C.J. et al. Prion strain discrimination using luminescent conjugated polymers. Nat. Meth. 4, 1023-1030
UniversitätsSpital Zürich, Institute of Neuropathology, Department of Pathology, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland.Nature Methods (Impact Factor: 32.07). 01/2008; 4(12):1023-30. DOI: 10.1038/nmeth1131
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). "
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.ILAR journal / National Research Council, Institute of Laboratory Animal Resources 05/2015; 56(1):7-25. DOI:10.1093/ilar/ilv008 · 2.39 Impact Factor
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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. "
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.ChemBioChem 03/2013; 14(5). DOI:10.1002/cbic.201200731 · 3.09 Impact Factor
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- "Binding to protein aggregates constrains the rotational freedom of the thiophene backbone, altering their spectral properties in a conformation-sensitive manner. Thus, an optical ‘fingerprint’ is obtained and this property has been used to discriminate prion protein aggregates associated with different prion strains [27,28], conformational heterogeneities in Amyloid-β amyloid plaques in Alzheimer disease mouse models , and morphologically different amyloid deposits in systemic amyloidoses . LCPs and LCOs have also proven useful for detection of disease associated protein aggregates that go undetected by ThT and Congo red [27,28]. "
ABSTRACT: Two different conformational isoforms or amyloid strains of insulin with different cytotoxic capacity have been described previously. Herein these filamentous and fibrillar amyloid states of insulin were investigated using biophysical and spectroscopic techniques in combination with luminescent conjugated oligothiophenes (LCO). This new class of fluorescent probes has a well defined molecular structure with a distinct number of thiophene units that can adopt different dihedral angles depending on its binding site to an amyloid structure. Based on data from surface charge, hydrophobicity, fluorescence spectroscopy and imaging, along with atomic force microscopy (AFM), we deduce the ultrastructure and fluorescent properties of LCO stained insulin fibrils and filaments. Combined total internal reflection fluorescence microscopy (TIRFM) and AFM revealed rigid linear fibrous assemblies of fibrils whereas filaments showed a short curvilinear morphology which assemble into cloudy deposits. All studied LCOs bound to the filaments afforded more blue-shifted excitation and emission spectra in contrast to those corresponding to the fibril indicating a different LCO binding site, which was also supported by less efficient hydrophobic probe binding. Taken together, the multi-tool approach used here indicates the power of ultrastructure identification applying AFM together with LCO fluorescence interrogation, including TIRFM, to resolve structural differences between amyloid states.International Journal of Molecular Sciences 12/2012; 13(2):1461-80. DOI:10.3390/ijms13021461 · 2.86 Impact Factor
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