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Multimodal fluorescene microscopy of prion strain specific PrP deposits stained by thiophene-bassed amyloid ligands
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
Department of Pathology, Unversity of California, San Diego, USA.
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2014 (English)In: Prion, ISSN 1933-6896, E-ISSN 1933-690X, Vol. 8, no 4, 319-329 p.Article in journal (Refereed) Published
Abstract [en]

The disease-associated prion protein (PrP) forms aggregates which vary in structural conformation yet share 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.

Place, publisher, year, edition, pages
Taylor & Francis, 2014. Vol. 8, no 4, 319-329 p.
National Category
Chemical Sciences Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-106792DOI: 10.4161/pri.29239ISI: 000348376000006OAI: oai:DiVA.org:liu-106792DiVA: diva2:719146
Available from: 2014-05-23 Created: 2014-05-23 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Luminescent molecular recognition of pathognomonic and aging associated protein aggregates
Open this publication in new window or tab >>Luminescent molecular recognition of pathognomonic and aging associated protein aggregates
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Various protein inclusions have been recognized to be associated with aging and pathogenic conditions, such as in Alzheimer’s disease, Parkinson’s disease, Type 2 diabetes, and the prionoses Creutzfeldt-Jakob disease, Chronic wasting disease (CWD), and Mad cow disease. The causative transition of protein aggregation is the alteration in the conformation of the protein that renders the protein susceptible towards self-assembly. Variations in the physico-chemical ultrastructure of the protein deposit, i.e. the conformation and the chemical nature of the fibril constituent protein monomers, translate into specific structure-property phenotype, hence clinicopathology. Upon transmission and/or propagation this phenomenon gives rise to specific protein aggregate strains. Today most potential treatments of the protein conformational diseases have been a huge failure, effectively due to late diagnosis and subsequent therapeutic intervention. An imperative for efficient treatment is early detection and accurate identification for proper clinical diagnosis.

The purpose of the studies in this thesis was to develop highly sensitive methods for detection and discrimination of age- and disease associated protein deposits both for in vitro and ex vivo utilization.

Herein we have shown that, for in vitro usage, Nile red will bind to amyloid-like protein aggregates derived from a plethora of precursor proteins. It was also found that the fluorescence was insensitive to acidic assay conditions in contrast to the standard in vitro dye Thioflavin T (ThT). Further, Nile red was shown to discriminate between conformational isoforms thus enabling conformational typing of amyloid structures.

For the development of ex vivo detection methods we employed luminescent conjugated oligothiophenes (LCOs) and utilized the structure-conformation induced optical properties of this class of protein aggregate ligands. The heptameric oligothiophene h-FTAA was successfully used to detect, with high sensitivity, protein deposits from various systemic amyloidoses (ATTR, AA, AL-λ/κ, and the local amyloidosis AIAPP) derived from biopsy specimens. Also aging-associated protein deposits were detected which was found promising for early detection of potentially pathogenic protein inclusions. Further, LCO staining of tissue sections was found compatible with immunolabeling enabling subtyping of involved proteins. Early detection of amyloidosis also requires relatively non-invasive methods, why h-FTAA staining was directed towards fine-needle-aspirated (FNA) abdominal fat tissue smears. Staining of protein deposits and detection with high sensitivity was also found in the fat tissue smears.

In addition to the relatively rare prionoses it has lately been shown that Alzheimer’s, Parkinson’s diseases share similar properties as the prion pathologies. Hence the urgent need for ligands that will recognize specific disease specific strain aggregates. Using an established murine model for prion strain propagation we were able to discriminate two different prion strains, murine adapted Sheep Scrapie (mSS) and murine adapted Chronic wasting disease (mCWD) from each other by using multimodal fluorescence microscopy entailing emission/excitation spectral imaging and fluorescent lifetime imaging (FLIM).

In conclusion we have shown that the LCOs will recognize protein aggregates with high sensitivity and selectivity. In addition we have shown that the LCOs detect protein aggregates that Congo red failed to recognize thus allowing potentially early diagnosis. Last, we show that the LCOs will recognize and discriminate between different protein aggregate strains which potentially will allow disease specific therapeutic targeting.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2014. 77 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1596
National Category
Chemical Sciences Natural Sciences
Identifiers
urn:nbn:se:liu:diva-106878 (URN)978-91-7519-334-2 (ISBN)
Public defence
2014-06-11, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 14:15 (Swedish)
Opponent
Supervisors
Available from: 2014-05-23 Created: 2014-05-23 Last updated: 2017-01-11Bibliographically approved
2. Anionic oligothiophenes: Optical tools for multimodal fluorescent assignment of protein aggregates
Open this publication in new window or tab >>Anionic oligothiophenes: Optical tools for multimodal fluorescent assignment of protein aggregates
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Luminescent conjugated oligothiophenes (LCOs) represent a useful and interesting class of materials well known for their abilities as transducers for colorimetric and fluorometric reporting. Specifically, they have the ability to produce a conformation-dependent spectral signature reflective of changes in their local environment.  This physical property makes conjugated polymers an indispensible tool in the toolbox of fluorescent reporters used for distinguishing protein aggregates. Because fluorescence measurements provide a number of parameters for observing changes within a system (e.g., changes in intensity, wavelength, energy transfer, and emission lifetime), the coupling of such measurements with the unique fluorescence reporting capabilities of LCOs has been successful in a number of biological systems. The Nilsson group has demonstrated the use of both polydisperse and monodisperse conjugated polythiophenes for the purpose of amyloid protein aggregate detection both in vitro and ex vivo. My doctoral studies have included synthesis and the photophysical evaluation of pentameric substituted oligothiophenes for utilization as molecular probes for investigating the structure and conformation of amyloid protein aggregates. Through the synthesis of a library of pentameric probes with variations in side-chain substituents, we have studied the effects of pH, solvent, and viscosity on probe behavior and spectral shifts to elucidate the role of chemical structure on probe performance. Through a clearer understanding of the nature of LCOs and their individual chromic responses, we hope to provide researchers and clinicians additional tools for investigating and “bringing to light” the multifaceted nature of amyloids.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2014. 41 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1630
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-111657 (URN)978-91-7519-205-5 (ISBN)
Public defence
2014-11-14, Visionen B-huset, Campus Valla, Linköpings universitet, Linköping, 09:15 (English)
Opponent
Supervisors
Available from: 2014-10-28 Created: 2014-10-28 Last updated: 2014-10-28Bibliographically approved
3. Poly-and oligothiophenes: Optical probes for multimodal fluorescent assessment of biological processes
Open this publication in new window or tab >>Poly-and oligothiophenes: Optical probes for multimodal fluorescent assessment of biological processes
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

One interesting class of molecules in the research field of imaging biological processes is luminescent conjugated polythiophenes, LCPs. These fluorescent probes have a flexible backbone consisting of repetitive thiophene units. Due to this backbone, the probes possess unique abilities to give rise to different spectral signatures depending on their target and environment. LCPs are a polydispersed material meaning there is an uneven distribution of lengths of the probe. Recently, monodispersed chemically well-defined material denoted luminescent conjugated oligothiophenes, LCOs, with an exact number of repetitive units and distinct sidechain functionalities along the backbone has been developed. LCOs have the advantages of being smaller which leads to higher ability to cross the blood brain barrier. The synthesis of minor chemical alterations is also more simplified due to the well-defined materials.

During my doctoral studies I have used both LCPs and LCOs to study biological processes such as conformational variation of protein aggregates in prion diseases and cellular uptake in normal cells and cancer cells. The research has generally been based on the probes capability to emit light upon irradiation and the interaction with their targets has mainly been assessed through variations in fluorescence intensity, emission-and excitation profiles and fluorescence lifetime decay. These studies verified the utility of LCPs and LCOs for staining and discrimination of both prion strains and cell phenotypes. The results also demonstrated the pronounced influence minor chemical modifications have on the LCO´s staining capacity.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2015. 54 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1693
National Category
Chemical Sciences Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-121815 (URN)10.3384/diss.diva-121815 (DOI)978-91-7685-986-5 (ISBN)
Public defence
2015-11-06, Planck, Fysikhuset, Campus Valla, Linköping, 10:15 (Swedish)
Opponent
Supervisors
Available from: 2015-10-07 Created: 2015-10-07 Last updated: 2015-10-07Bibliographically approved

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Magnusson, KarinSimon, RozalynSjölander, DanielHammarström, PerNilsson, Peter R

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