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Sandberg, A. (2019). Polymorphic protein aggregation in tauopathies. (Doctoral dissertation). Linköping: Linköping University Electronic Press
Open this publication in new window or tab >>Polymorphic protein aggregation in tauopathies
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Alzheimer’s disease(s) comprises one of the most common and costly neurodegenerative diseases. With a larger population and an increasing life expectancy, amyloid diseases (with age as one of the most prominent risk factors) will generate an even larger burden on healthcare. We know that protein misfolding is involved in the disease process but lack a complete understanding of the mechanism behind these diseases, both the sporadic and hereditary variants. It is not always known whether it is a gain-of-toxic function or loss‐of‐function that causes the neurodegeneration. To determine the correct diagnosis is a major challenge. If diagnosed, only a few amyloid diseases can be treated today.

Amyloids are highly ordered filamentous protein aggregates with a β‐sheet structure. From identical or similar amino acid sequences, a large variety of structures can be formed by different secondary and tertiary structures and by different packing of the individual filaments. This is known as fibril polymorphism.

This work focuses on characterization on two proteins involved in Alzheimer’s disease and other neurodegenerative diseases, namely Amyloid‐β (Aβ) and microtubule associated protein tau (tau). In order to investigate the properties of these proteins in vitro it is important to have protocols for production of recombinant protein that enables characterization of these aggregation prone proteins. We present protocols for recombinant expression, purification and non‐denaturing fibrillation assays used in our lab to produce and analyze Aβ, tau and the prion protein.

Development of new ligands for characterization of fibrils is an important way of investigating different fibrillary structures and characterizing and distinguishing between the different polymorphs of aggregates. We showed that the central benzene ring of the amyloid ligand X‐34 can be exchanged for other heterocyclic motifs and still retain targeting of the “Congo red” binding site. The compounds do not compete with the Pittsburgh compound B (PiB) binding site on recombinant Aβ fibrils.

We also characterized tau fibrils formed from seeding with tau aggregates from patients diagnosed with different neurodegenerative tauopathies. We use aggregation kinetics to test the seeding activity on two different sequence isoforms of tau, 0N3R and 0N4R. Fibrillation kinetics, an array of recently developed ligands (including the X‐34 analogs) and electron microscopy were used to characterize different polymorphs of the tau aggregates formed by seeded templating from patient derived seeds. Our data showed that brains contain seeds with different morphologies even with in patients diagnosed with the same disease.

Investigations of the rare tau mutant G273R found in a patient with a presumed tauopathy also highlights the problem with proper diagnostics. Our results reveal that in vitro this mutation change the binding properties of 0N4R tau to the cytoskeletal proteins microtubule and F‐actin. Furthermore, we could show that when seeded, the fibril formation seeding activity followed a sequence similarity dependent manner. In fibrils formed during heparin-induced aggregation we can be distinguished between wild type and mutant tau as they form fibrils with different thickness. Our in vitro biophysical data support that the G237R mutant is causing a 4R tauopathy.

The work in this thesis increase our knowledge in the field of tau aggregation and tau fibril polymorphism.

Abstract [sv]

En av de vanligaste och mest kostsamma sjukdomarna är den nervdödande Alzheimers sjukdom. Med en större population och ökad förväntad livslängd kommer amyloida sjukdomar, som har ålder som den viktigaste riskfaktorn, att generera en ökad börda för sjukvården. Vi saknar en fullständig förståelse för mekanismerna bakom dessa sjukdomar både för de sporadiska och ärftliga varianterna. Man vet att felveckade proteiner är inblandade i dessa sjukdomar. Det är inte alltid känt hur den felveckade formen av ett protein alstrar en toxisk funktion eller om det är en förlust av dennas funktion som orsakar nervdöden. Att kunna fastställa en korrekt diagnos är en stor utmaning för forskarvärlden idag. Även när en korrekt diagnos kan ställas är det endast ett fåtal amyloida sjukdomar som kan behandlas idag.

Amyloider är mycket välordnade filamentösa proteinaggregat med β-flakstruktur. Från identiska eller liknande aminosyrasekvenser kan ett stort antal strukturer bildas med olika sekundär- och tertiär struktur och olika packning av individuella filament. Vi kallar detta för strukturell polymorfism.

Det här arbetet fokuserar på karakterisering av två proteiner involverade i Alzheimers sjukdom och andra neurodegenerativa sjukdomar nämligen Amyloid β (Aβ) och mikrotubuli associerade protein tau (tau). För att kunna undersöka egenskaperna hos dessa proteiner är det viktigt att ha protokoll för produktion av rekombinant protein för att kunna karakterisera dessa aggregeringsbenägna proteiner. Vi utvecklade protokoll för rekombinant utryck, rening och icke-denaturerande fibrilleringsanalyser som används i vårt labb för att producera och analysera Aβ, tau och prionproteinet.

Utveckling av nya ligander för karakterisering av fibriller är en viktig väg för att undersöka olika fibrillstrukturer och för karakterisering och för att kunna särskilja mellan olika polymorfer av aggregat. I det här arbetet visas att den centrala bensenringen hos amyloidliganden X-34 kan bytas ut mot andra heterocykliska motiv och fortfarande behålla sin specificitet mot ”Congo röd” bindnings-sätet utan att konkurrera med Pittsburgh compound B (PiB) bindnings-säte på rekombinanta Aβ fibriller.

Vi karaktäriserade också tau fibriller bildade via ympning, så kallad seeding, med tau aggregat isolerade från patienter diagnosticerade med olika nervdödande taupatier. Vi använder aggregerings kinetik för att testa seedningsförmåga på två olika sekvens isoformer av tau. Nyligen utvecklade ligander (inkluderat X-34 analoger) och elektronmikroskopi användes för att karakterisera de olika polymorferna av tau aggregaten. Våra data påvisar att olika patienter bär på olika seeds, det vill säga olika polymorpher. Även mellan patienter med samma diagnos finns skillnader.

Undersökningar av den ovanliga tau mutationen G273R understryker också problemet med fastställandet av korrekt diagnos. Våra resultat från provrörsexperiment avslöjar att den här mutationen ändrar bindningsegenskaperna av 0N4R tau till cytoskelettproteinerna mikrotubulin och F-aktin. Vi kunde ytterligare visa att när fibrilleringsreaktionen seedades så följde det en sekvenslikhetsberoende mekanism. Fibrerna som bildas under heparininducering kan skiljas åt mellan normalt och muterat tau genom att de har olika tjocklek. Våra biofysikaliska data stödjer att G273R tau mutationen kan orsaka en 4R tauopati.

Arbetet i denna avhandling ökar vår kunskap inom området tau-aggregering och tau fibrilpolymorfism.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2019. p. 51
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 2022
National Category
Biophysics Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-161659 (URN)10.3384/diss.diva-161659 (DOI)9789179299842 (ISBN)
Public defence
2019-12-20, Planck, Physics Building, Campus Valla, Linköping, 09:00 (English)
Opponent
Supervisors
Available from: 2019-11-08 Created: 2019-11-07 Last updated: 2019-12-12Bibliographically approved
Zhang, J., Wang, J., Sandberg, A., Wu, X., Nyström, S., LeVine, H. I., . . . Lindgren, M. (2018). Intramolecular Proton and Charge Transfer of Pyrene-based trans-Stilbene Salicylic Acids Applied to Detection of Aggregated Proteins.. ChemPhysChem, 19(22), 3001-3009
Open this publication in new window or tab >>Intramolecular Proton and Charge Transfer of Pyrene-based trans-Stilbene Salicylic Acids Applied to Detection of Aggregated Proteins.
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2018 (English)In: ChemPhysChem, ISSN 1439-4235, E-ISSN 1439-7641, Vol. 19, no 22, p. 3001-3009Article in journal (Refereed) Published
Abstract [en]

Two analogues to the fluorescent amyloid probe 2,5-bis(4'-hydroxy-3'-carboxy-styryl)benzene (X-34) were synthesized based on the trans-stilbene pyrene scaffold (Py1SA and Py2SA). The compounds show strikingly different emission spectra when bound to preformed Aβ1-42 fibrils. This remarkable emission difference is retained when bound to amyloid fibrils of four distinct proteins, suggesting a common binding configuration for each molecule. Density functional theory calculations show that Py1SA is twisted, while Py2SA is more planar. Still, an analysis of the highest occupied molecular orbitals (HOMOs) and lowest unoccupied molecular orbitals (LUMOs) of the two compounds indicates that the degree of electronic coupling between the pyrene and salicylic acid (SA) moieties is larger in Py1SA than in Py2SA. Excited state intramolecular proton transfer (ESIPT) coupled-charge transfer (ICT) was observed for the anionic form in polar solvents. We conclude that ICT properties of trans-stilbene derivatives can be utilized for amyloid probe design with large changes in emission spectra and decay times from analogous chemical structures depending on the detailed physical nature of the binding site.less thanbr /greater than (© 2018 Wiley-VCH Verlag GmbH and Co. KGaA, Weinheim.)

Place, publisher, year, edition, pages
Weinheim, Germany: Wiley-VCH Verlag, 2018
National Category
Theoretical Chemistry
Identifiers
urn:nbn:se:liu:diva-152767 (URN)10.1002/cphc.201800823 (DOI)000450672100006 ()30183138 (PubMedID)
Available from: 2018-11-20 Created: 2018-11-20 Last updated: 2019-11-08
Sandberg, A. & Nyström, S. (2018). Purification and Fibrillation of Recombinant Human Amyloid-ß, Prion Protein, and Tau Under Native Conditions. In: Einar M. Sigurdsson, Miguel Calero and María Gasset (Ed.), Amyloid Proteins: Methods and Protocols (pp. 147-166). Humana Press, 1779
Open this publication in new window or tab >>Purification and Fibrillation of Recombinant Human Amyloid-ß, Prion Protein, and Tau Under Native Conditions
2018 (English)In: Amyloid Proteins: Methods and Protocols / [ed] Einar M. Sigurdsson, Miguel Calero and María Gasset, Humana Press, 2018, Vol. 1779, p. 147-166Chapter in book (Refereed)
Abstract [en]

Protein misfolding, aggregation, and amyloid formation is involved in a large number of diseases. Recombinantly expressed proteins to study the amyloid fibril formation process are important for mechanistic studies. We here report protocols for production, purification, and fibrillation of three different proteins commonly found in cerebral amyloid; Aß and Tau found in Alzheimers disease, Chronic traumatic brain injury, Corticobasal degeneration, and Progressive Supranuclear Palsy and human prion protein found in Creutzfeldt-Jakobs disease. The three protocols have in common that the protein is in a pH-neutral phosphate saline buffer during fibrillation to mimic their endogenous near physiological environment.

Place, publisher, year, edition, pages
Humana Press, 2018
Series
Methods in Molecular Biology, E-ISSN 1940-6029 ; 1779
Keywords
Amyloid; Aß; Fibrillation; Neurodegenerative disease; Prion protein; Purification; Recombinant; Tau
National Category
Clinical Laboratory Medicine
Identifiers
urn:nbn:se:liu:diva-152517 (URN)10.1007/978-1-4939-7816-8_10 (DOI)29886532 (PubMedID)9781493978151 (ISBN)9781493978168 (ISBN)
Available from: 2019-03-28 Created: 2019-03-28 Last updated: 2019-11-08
Zhang, J., Sandberg, A., Wu, X., Nyström, S., Lindgren, M., Konradsson, P. & Hammarström, P. (2017). trans-Stilbenoids with Extended Fluorescence Lifetimes for the Characterization of Amyloid Fibrils. ACS Omega, 2(8), 4693-4704
Open this publication in new window or tab >>trans-Stilbenoids with Extended Fluorescence Lifetimes for the Characterization of Amyloid Fibrils
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2017 (English)In: ACS Omega, ISSN 2470-1343, Vol. 2, no 8, p. 4693-4704Article in journal (Refereed) Published
Abstract [en]

It was previously reported that two naphthyl-based trans-stilbene probes, (E)-4-(2-(naphthalen-1-yl)vinyl)benzene-1,2-diol (1) and (E)-4-(2-(naphthalen-2-yl)vinyl)benzene-1,2-diol (3), can bind to both native transthyretin (TTR) and misfolded protofibrillar TTR at physiological concentrations, displaying distinct emission maxima bound to the different conformational states (>100 nm difference). To further explore this amyloid probe scaffold to obtain extended fluorescence lifetimes, two new analogues with expanded aromatic ring systems (anthracene and pyrene), (E)-4-(2-(anthracen-2-yl)vinyl)benzene-1,2-diol (4) and (E)-4-(2-(pyren-2-yl)vinyl)benzene-1,2-diol (5), were synthesized employing the palladium-catalyzed Mizoroki–Heck reaction. (E)-4-Styrylbenzene-1,2-diol (2), 3, 4, and 5 were investigated with respect to their photophysical properties in methanol and when bound to insulin, lysozyme, and Aβ1-42 fibrils, including time-resolved fluorescence measurements. In conclusion, 4 and 5 can bind to both native and fibrillar TTR, becoming highly fluorescent. Compounds 2–5 bind specifically to insulin, lysozyme, and Aβ1-42 fibrils with an apparent fluorescence intensity increase and moderate binding affinities. The average fluorescence lifetimes of the probes bound to Aβ1-42 fibrils are 1.3 ns (2), 1.5 ns (3), 5.7 ns (4), and 29.8 ns (5). In summary, the variable aromatic moieties of the para-positioned trans-stilbenoid vinyl-benzene-1,2-diol with benzene, naphthalene, anthracene, and pyrene showed that the extended conjugated systems retained the amyloid targeting properties of the probes. Furthermore, both the anthracene and pyrene moieties extensively enhanced the fluorescence intensity and prolonged lifetimes. These attractive probe properties should improve amyloid detection and characterization by fluorescence-based techniques.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2017
Keywords
Fluorescence; Glycoproteins; Molecular association; Molecular recognition; Optical materials; Quantum transition; Spectra
National Category
Organic Chemistry
Identifiers
urn:nbn:se:liu:diva-151658 (URN)10.1021/acsomega.7b00535 (DOI)000409924000069 ()
Available from: 2018-09-28 Created: 2018-09-28 Last updated: 2019-11-08Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-6364-2690

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