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Luminescent molecular recognition of pathognomonic and aging associated protein aggregates
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
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: urn:nbn:se:liu:diva-106878ISBN: 978-91-7519-334-2 (print)OAI: oai:DiVA.org:liu-106878DiVA: diva2:719163
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
List of papers
1. Spectroscopic characterization of diverse amyloid fibrils in vitro by the fluorescent dye Nile red
Open this publication in new window or tab >>Spectroscopic characterization of diverse amyloid fibrils in vitro by the fluorescent dye Nile red
2011 (English)In: MOLECULAR BIOSYSTEMS, ISSN 1742-206X, Vol. 7, no 4, 1232-1240 p.Article in journal (Refereed) Published
Abstract [en]

The fluorescence of Nile red (9-diethylamino-5H-benzophenoxazine-5-one) is quenched in aqueous solutions but shows augmented fluorescence in hydrophobic environments. Nile red fluorescence was blue shifted and strongly augmented in the presence of various amyloid fibrils assayed under acidic as well as neutral pH conditions. Fibrils grown from lysozyme and insulin (at pH 1.6 and 65 degrees C), transthyretin (TTR) fibrils grown from the acid unfolded monomer (pH 2.0, 21 degrees C) or from the dissociated tetramer starting from native protein under less acidic conditions (pH 4.4, 37 degrees C) were detected. Nile red was also successfully employed in detecting A beta 1-42 and human prion protein (PrP90-231) amyloid fibrils grown at neutral pH. Nile red was amyloid fibril specific and did not fluoresce appreciably in the presence of the monomeric precursor proteins. Stokes shifts of the wavelength maximum of Nile red bound to various fibrils were different (ranging from 615 nm to 638 nm) indicating sensitivity to the tertiary structure in its respective binding sites of different amyloid proteins. A polarity assay using ethanol-water mixtures and pure octanol ranging from dielectric constants between 10 and 70 showed a linear correlation of Nile red Stokes shift and allowed assignment of amyloid fibril binding site polarity. Fluorescence resonance energy transfer between Thioflavin T (ThT) and Nile red was proven to be efficient and co-staining was employed to discriminate between conformational isoforms of A beta 1-42 amyloid fibrils grown under agitated and quiescent conditions. This paper demonstrates the complementary use of this fluorometric method for conformational typing of amyloid structures.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2011
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-67157 (URN)10.1039/c0mb00236d (DOI)000288329300031 ()
Available from: 2011-04-01 Created: 2011-04-01 Last updated: 2015-05-28
2. Luminescent conjugated oligothiophenes: A novel dye for amyloid diagnostics
Open this publication in new window or tab >>Luminescent conjugated oligothiophenes: A novel dye for amyloid diagnostics
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2013 (English)In: XIIIth International Symposium on Amyloidosis: From Misfolded Proteins to Well-Designed Treatment: The Proceedings of the XIIIth International Symposium on Amyloidosis,May 6-10, 2012, Groningen, The Netherlands / [ed] Bouke P.C. Hazenberg and Johan Bijzet, GUARD (Groningen Unit for Amyloidosis Research & Development) , 2013, 179-182 p.Conference paper, Published paper (Refereed)
Abstract [en]

The alkaline Congo red staining method has, for almost half a century, been the gold standard of amyloid diagnosis. Unfortunately, the method is both laborious and requires great skill to achieve proper diagnosis. In this study we are presenting an alternative method that is compatible with immunofluorescence typing. We used a novel dye, h-FTAA, designed and synthesized by us. The dye belongs to the novel class of conformation sensitive dyes known as Luminescent conjugated oligothiophenes (LCOs). We examined 37 different cases of systemic amyloidoses from various tissues. It was found that h-FTAA binds to amyloid with higher sensitivity and greater selectivity than Congo red, as was determined by both fluorescence- and light polarization microscopy. Due to the methods ease of use and performance compared to Congo red, it is concluded that h-FTAA is a better first choice for screening of systemic amyloidoses.

Place, publisher, year, edition, pages
GUARD (Groningen Unit for Amyloidosis Research & Development), 2013
National Category
Chemical Sciences Natural Sciences
Identifiers
urn:nbn:se:liu:diva-106789 (URN)978-90-821593-0-1 (ISBN)978-90-821593-1-8 (ISBN)
Conference
The XIIIth International Symposium on Amyloidosis, May 6-10, 2012, Groningen, The Netherlands
Available from: 2014-05-23 Created: 2014-05-23 Last updated: 2014-05-23Bibliographically approved
3. Evaluation of the fluorescent amyloid ligand h-FTAA in human tissues with systemic and localized amyloid
Open this publication in new window or tab >>Evaluation of the fluorescent amyloid ligand h-FTAA in human tissues with systemic and localized amyloid
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2014 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Rapid and accurate detection of amyloid deposits in routine surgical pathology settings are of great importance. The use of fluorescence microscopy in combination with appropriate amyloid specific dyes is very promising in this regard. Most systemic amyloidosis are progressive and lethal. Disease specific therapy depends on the identification of the offending proteins. Here we report that a luminescent conjugated oligothiophene, h-FTAA, rapidly and with high sensitivity and selectivity detects amyloid deposits in verified clinical samples from systemic amyloidosis patients with AA, AL, and ATTR types; as well as in tissues laden with localized amyloidosis of AANF, AIAPP and ASem1 type. The probe h-FTAA emitted yellow red fluorescence on binding to amyloid deposits, whereas no apparent staining was observed in surrounding tissue. Screening of 114 amyloid containing tissues derived from §07 verified (Congo red birefringence and immunohistochemistry) amyloidosis patients revealed complete correlation between h-FTAA and Congo red fluorescence. We conclude that h-FTAA is a fluorescent hypersensitive, rapid and powerful tool for identifying amyloid deposits in tissue sections. H-FTAA staining can be utilized as a rapid complementary technique for accurate detection of amyloid in routine surgical pathology settings. It was also revealed that within 5 of 15 age matched Congo red negative control samples h-FTAA detects microdeposits of amyloid-like protein aggregates in liver and kidney. The results emphasize the potential of the dye for detection of prodromal amyloidosis as well as for discovery of novel amyloid-like protein aggregates in humans.

National Category
Chemical Sciences Natural Sciences
Identifiers
urn:nbn:se:liu:diva-106790 (URN)
Available from: 2014-05-23 Created: 2014-05-23 Last updated: 2014-05-23Bibliographically approved
4. Sensitive and rapid assessment of amyloid by oligothiophene fluorescence in subcutaneous fat tissue
Open this publication in new window or tab >>Sensitive and rapid assessment of amyloid by oligothiophene fluorescence in subcutaneous fat tissue
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2015 (English)In: Amyloid: Journal of Protein Folding Disorders, ISSN 1350-6129, E-ISSN 1744-2818, Vol. 22, no 1, 19-25 p.Article in journal (Refereed) Published
Abstract [en]

Systemic amyloidosis (SA) is often diagnosed late. Combining clinical and biochemical biomarkers is necessary for raising suspicion of disease. Fine needle aspiration (FNA) of subcutaneous fat enables SA detection by Congo red staining. The luminescent conjugated probe heptameric formic thiophene acetic acid (h-FTAA) is a sensitive alternative to Congo red-staining of tissue samples. Our objective was to compare h-FTAA fluorescence with the Congo red stain for amyloid detection in FNA-obtained fat tissue. Herein, we studied samples from 57 patients with established SA (19 with AA, 20 with AL, and 18 with ATTR) and 17 age-matched controls (34–75 years). Positivity for h-FTAA was graded according to a Congo red-based grading scale ranging from 0 to 4+. Amyloid grading by both methods correlated strongly (r = 0.87). Here h-FTAA was positive in 53 of 54 Congo red-positive cases (sensitivity 98%) and h-FTAA was negative in 7 of 17 Congo red-negative controls (specificity 41%), but was also positive for 3 Congo red-negative SA cases. We conclude that h-FTAA fluorescence is more sensitive than Congo red staining in this small exploratory study of fat tissue samples, implicating potential sensitivity for prodromal amyloidosis, but is less specific for clinical amyloidosis defined by Congo red positivity. Given its simplicity h-FTAA staining may therefore be the most appropriate method for rapid screening of fat tissue samples but should presently treat grade 1+ as only suggestive, whereas 2+ or higher as positive for amyloidosis. Parallel assessment of h-FTAA and Congo red staining appears highly promising for clinical applications.

Place, publisher, year, edition, pages
Informa Healthcare, 2015
Keyword
AA amyloidosis, amyloid light chain, hyper spectral imaging, systemic amyloidosis, transthyretin
National Category
Chemical Sciences
Identifiers
urn:nbn:se:liu:diva-117806 (URN)10.3109/13506129.2014.984063 (DOI)000352637800003 ()25847117 (PubMedID)
Note

At the time for thesis presentation publication was in status: Manuscript

Funding Agencies|European Commission; Swedish Foundation for Strategic Research; Swedish Research Council; Linkoping University Center for Neuroscience; European Research Council

Available from: 2015-05-11 Created: 2015-05-08 Last updated: 2017-12-04Bibliographically approved
5. Multimodal fluorescene microscopy of prion strain specific PrP deposits stained by thiophene-bassed amyloid ligands
Open this publication in new window or tab >>Multimodal fluorescene microscopy of prion strain specific PrP deposits stained by thiophene-bassed amyloid ligands
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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
National Category
Chemical Sciences Natural Sciences
Identifiers
urn:nbn:se:liu:diva-106792 (URN)10.4161/pri.29239 (DOI)000348376000006 ()
Available from: 2014-05-23 Created: 2014-05-23 Last updated: 2017-12-05Bibliographically approved
6. Enhanced Fluorescent Assignment of Protein Aggregates by an Oligothiophene-Porphyrin-Based Amyloid Ligand
Open this publication in new window or tab >>Enhanced Fluorescent Assignment of Protein Aggregates by an Oligothiophene-Porphyrin-Based Amyloid Ligand
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2013 (English)In: Macromolecular rapid communications, ISSN 1022-1336, E-ISSN 1521-3927, Vol. 34, no 9, 723-730 p.Article in journal (Refereed) Published
Abstract [en]

Fluorescent probes identifying protein aggregates are of great interest, as deposition of aggregated proteins is associated with many devastating diseases. Here, we report that a fluorescent amyloid ligand composed of two distinct molecular moieties, an amyloidophilic pentameric oligothiophene and a porphyrin, can be utilized for spectral and lifetime imaging assessment of recombinant A 1-42 amyloid fibrils and A deposits in brain tissue sections from a transgenic mouse model with Alzheimers disease pathology. The enhanced spectral range and distinct lifetime diversity of this novel oligothiopheneporphyrin-based ligand allow a more precise assessment of heterogeneous amyloid morphology compared with the corresponding oligothiophene dye.

Place, publisher, year, edition, pages
Wiley-VCH Verlag, 2013
Keyword
oligothiophene, porphyrin, protein deposits, imaging, fluorescence
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-93385 (URN)10.1002/marc.201200817 (DOI)000318354500004 ()
Note

Funding Agencies|Swedish Research Council||Knut and Alice Wallenberg Foundation||Swedish Foundation for Strategic Research||European Union FP7 HEALTH (Project LUPAS)||LiU Neuroscience Center||ERC Starting Independent Researcher grant (Project: MUMID)||

Available from: 2013-05-31 Created: 2013-05-31 Last updated: 2017-12-06

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