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Fluorescent thiophene-based ligands for detection and characterization of disease-associated protein aggregates
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, The Institute of Technology.
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis the unique optical properties of fluorescent ligands termed luminescent conjugated oligothiophenes (LCOs) have been used to study a variety of protein aggregates associated with human protein misfolding disease. This heterogeneous group of diseases contains well known and fatal members such as Alzheimer´s and Huntington´s disease and the development of sensitive tools for the detection and characterization of protein aggregates is crucial for unravelling the complexity of these pathologies. Conventionally, the molecular dyes Congo red and thioflavin T (ThT) have been the primary choices for detecting and monitoring protein misfolding events. However, the rigid scaffold of both Congo red and ThT only offers an on or off mode and limits their ability to make fine distinctions at the molecular level. In contrast, LCOs have a flexible conjugated backbone and in addition to detect a broader subset of misfolded proteins, LCO can be used to visualize the heterogeneity of protein aggregates.

The work presented in this thesis has given novel insights regarding the close connection between LCO design and optical performance. By altering the backbone length and the arrangement of substituents as well as replacing thiophene units with moieties affecting conjugation length and conformational freedom, the structural requirements of an optimal LCO for a certain application have been revealed. LCOs having a pentameric thiophene backbone with carboxyl end-groups were able to i) cross the blood-brain barrier and selectively stain cerebral amyloid β (Aβ) plaques, ii) detect non-thioflavinophilic Aβ aggregates and non-congophilic prion aggregates, iii) spectrally discriminate Aβ from tau aggregates and iiii) strongly label protein inclusion bodies. However, in some applications this design was outdone by others and in general, the conjugation length and the level of conformational freedom of the backbone were important determinants of the performance of the LCO.

Overall, the findings in this thesis illustrate how small alterations in the LCO molecular scaffold may have large impact on the ligand properties. The results highlight the importance of having a toolbox of diverse ligands in order to increase our knowledge regarding the complex nature of protein aggregates.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2013. , 81 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1518
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-92772ISBN: 978-91-7519-623-7 (print)OAI: oai:DiVA.org:liu-92772DiVA: diva2:622391
Public defence
2013-05-31, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:15 (Swedish)
Opponent
Supervisors
Available from: 2013-05-21 Created: 2013-05-21 Last updated: 2014-04-08Bibliographically approved
List of papers
1. Novel Pentameric Thiophene Derivatives for in Vitro and in Vivo Optical Imaging of a Plethora of Protein Aggregates in Cerebral Amyloidoses
Open this publication in new window or tab >>Novel Pentameric Thiophene Derivatives for in Vitro and in Vivo Optical Imaging of a Plethora of Protein Aggregates in Cerebral Amyloidoses
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2009 (English)In: ACS CHEMICAL BIOLOGY, ISSN 1554-8929, Vol. 4, no 8, 673-684 p.Article in journal (Refereed) Published
Abstract [en]

Molecular probes for selective Identification of protein aggregates are important to advance our understanding of the molecular pathogenesis underlying cerebral amyloidoses. Here we report the chemical design of pentameric thiophene derivatives, denoted luminescent conjugated oligothiophenes (LCOs), which could be used for real-time visualization of cerebral protein aggregates in transgenic mouse models of neurodegenerative diseases by multiphoton microscopy. One of the LCOs, p-FTAA, could be utilized for ex vivo spectral assignment of distinct prion deposits from two, mouse-adapted prion strains. p-FTAA also revealed a transient soluble pre-fibrillar non-thioflavinophilic A beta-assemblies during in vitro fibrillation of A beta peptides. In brain tissue samples, A beta deposits and neurofibrillary tangles (NFTs) were readily identified by a strong fluorescence from p-FTAA and the LCO staining showed complete co-localliation with conventional antibodies (6E10 and AT8). In addition, a patchy islet-like staining of individual A beta plaque was unveiled by the anti-oligomer A11 antibody during co-staining with p-FTAA. The major hallmarks of Alzheimers disease, namely, A beta aggregates versus NFTs, could also be distinguished because of distinct emission spectra from p-FTAA. Overall, we demonstrate that LCOs can be utilized as powerful practical research tools for studying protein aggregation diseases and facilitate the study of amyloid origin, evolution and maturation, A beta-tau interactions, and pathogenesis both ex vivo and in vivo.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-20420 (URN)10.1021/cb900112v (DOI)
Available from: 2009-09-08 Created: 2009-09-07 Last updated: 2015-05-28
2. Synthesis of a library of oligothiophenes and their utilization as fluorescent ligands for spectral assignment of protein aggregates
Open this publication in new window or tab >>Synthesis of a library of oligothiophenes and their utilization as fluorescent ligands for spectral assignment of protein aggregates
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2011 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 9, no 24, 8356-8370 p.Article in journal (Refereed) Published
Abstract [en]

Molecular probes for selective identification of protein aggregates are important to advance our understanding of the molecular pathogenesis underlying protein aggregation diseases. Here we report the chemical design of a library of anionic luminescent conjugated oligothiophenes (LCOs), which can be utilized as ligands for detection of protein aggregates. Certain molecular requirements were shown to be necessary for detecting (i) early non-thioflavinophilic protein assemblies of A beta 1-42 and insulin preceding the formation of amyloid fibrils and (ii) for obtaining distinct spectral signatures of the two main pathological hallmarks observed in human Alzheimers diease brain tissue (A beta plaques and neurofibrillary tangles). Our findings suggest that a superior anionic LCO-based ligand should have a backbone consisting of five to seven thiophene units and carboxyl groups extending the conjugated thiophene backbone. Such LCOs will be highly useful for studying the underlying molecular events of protein aggregation diseases and could also be utilized for the development of novel diagnostic tools for these diseases.

Place, publisher, year, edition, pages
Royal Society of Chemistry, 2011
National Category
Organic Chemistry
Identifiers
urn:nbn:se:liu:diva-73487 (URN)10.1039/c1ob05637a (DOI)000297354100019 ()
Available from: 2012-01-04 Created: 2012-01-04 Last updated: 2017-12-08
3. The structural basis for optimal performance of oligothiophene based fluorescent amyloid ligands: Conformational flexibility is essential for spectral assignment of a diversity of protein aggregates
Open this publication in new window or tab >>The structural basis for optimal performance of oligothiophene based fluorescent amyloid ligands: Conformational flexibility is essential for spectral assignment of a diversity of protein aggregates
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Protein misfolding diseases are characterized by deposition of protein aggregates and optical ligands for molecular characterization of these disease-associated structures are important for understanding their potential role in the pathogenesis of the disease. Luminescent conjugated oligothiophenes (LCOs) have proven useful for optical identification of a broader subset of disease-associated protein aggregates than conventional ligands, such as Thioflavin T (ThT) and Congo red. Herein, the molecular requirements for achieving LCOs able to detect non-thioflavinophilic Aβ aggregates or non-congophilic prion aggregates, as well as spectrally discriminate Aβ and tau aggregates, were investigated. An anionic pentameric LCO was subjected to chemical engineering by i) replacing thiophene units with selenophene or phenylene moieties or ii) alternating the anionic substituents along the  thiophene backbone. In addition, two asymmetric tetrameric ligands were  generated. Overall, the results from this study identified conformational  freedom and extended conjugation of the conjugated backbone as crucial  determinants for obtaining superior thiophene-based optical ligands for  sensitive detection and spectral assignment of diseaseassociated protein aggregates.

Keyword
Luminescent conjugated oligothiophenes, protein aggregates, fluorescence, Alzheimer´s disease, prion
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-92771 (URN)
Available from: 2013-05-21 Created: 2013-05-21 Last updated: 2014-04-08
4. Cross beta-Sheet Conformation of Keratin 8 Is a Specific Feature of Mallory-Denk Bodies Compared With Other Hepatocyte Inclusions
Open this publication in new window or tab >>Cross beta-Sheet Conformation of Keratin 8 Is a Specific Feature of Mallory-Denk Bodies Compared With Other Hepatocyte Inclusions
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2011 (English)In: Gastroenterology, ISSN 0016-5085, E-ISSN 1528-0012, Vol. 141, no 3, 1080-U428 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND andamp; AIMS: Mallory-Denk bodies (MDBs) are cytoplasmic protein aggregates in hepatocytes in steato-hepatitis and other liver diseases. We investigated the molecular structure of keratin 8 (K8) and 18 (K18), sequestosome 1/p62, and ubiquitin, which are the major constituents of MDBs, to investigate their formation and role in disease pathogenesis. METHODS: Luminescent conjugated oligothiophenes (LCOs), h-HTAA, and p-FTAA are fluorescent amyloid ligands that specifically bind proteins with cross beta-sheet conformation. We used LCOs to investigate conformational changes in MDBs in situ in human and murine livers as well as in transfection studies. RESULTS: LCO analysis showed cross beta-sheet conformation in human MDBs from patients with alcoholic and nonalcoholic steatohepatitis or hepatocellular carcinoma, but not in intracellular hyaline bodies, alpha(1)-antitrypsin deficiency, or ground-glass inclusions. LCOs bound to MDBs induced by 3,5diethoxycarbonyl-1,4-dihydrocollidine feeding of mice at all developmental stages. CHO-K1 cells transfected with various combinations of SQSTM1/p62, ubi, and Krt8/Krt18 showed that K8 was more likely to have cross beta-sheet conformation than K18, whereas p62 never had cross beta-sheet conformation. The different conformational properties of K8 and K18 were also shown by circular dichroism analysis. CONCLUSIONS: K8 can undergo conformational changes from predominantly alpha-helical to cross beta-sheet, which would allow it to form MDBs. These findings might account for the observation that krt8(-/-) mice do not form MDBs, whereas its excess facilitates MDB formation. LCOs might be used in diagnosis of liver disorders; they can be applied to formalin-fixed, paraffin-embedded tissues to characterize protein aggregates in liver cells.

Place, publisher, year, edition, pages
WB Saunders, 2011
Keyword
Liver Disease, Protein Aggregation, Keratin, p62, NASH
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-70739 (URN)10.1053/j.gastro.2011.05.039 (DOI)000294281200047 ()
Note

Funding Agencies|Austrian Genome Program (GEN-AU)||Medical University of Graz||Swedish Foundation for Strategic Research||Knut and Alice Wallenberg Foundation||

Available from: 2011-09-16 Created: 2011-09-16 Last updated: 2017-12-08
5. Luminescent Conjugated Oligothiophenes for Sensitive Fluorescent Assignment of Protein Inclusion Bodies
Open this publication in new window or tab >>Luminescent Conjugated Oligothiophenes for Sensitive Fluorescent Assignment of Protein Inclusion Bodies
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2013 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 14, no 5, 607-616 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Wiley-VCH Verlag Berlin, 2013
Keyword
amyloid beta-peptides, biosensors, fluorescent probes, luminescent conjugated oligothiophene, protein inclusion body
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-91341 (URN)10.1002/cbic.201200731 (DOI)000316285600010 ()
Note

Funding Agencies|Swedish Foundation for Strategic Research||European Research Council (ERC)||EU||

Available from: 2013-04-23 Created: 2013-04-22 Last updated: 2017-12-06Bibliographically approved

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