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Investigating Amyloid β toxicity in Drosophila melanogaster
Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis Drosophila melanogaster (the fruit fly) has been used as a model organism to study the aggregation and toxic properties of the human amyloid β (Aβ) peptide involved in the onset of Alzheimer's disease (AD). AD is one of many misfolding diseases where the important event of a protein to adopt its’ specific three-dimensional structure has failed, leading to aggregation and formation of characteristic amyloid fibrils. AD has a complex pathology and probably reflects a variety of related molecular and cellular abnormalities, however, the most apparent common denominator so far is abnormal Amyloid-β precursor protein (APP) processing, resulting in a pool of various Aβ-peptides. In AD, the Aβ peptide misfolds, aggregates and forms amyloid plaques in the brain of patients, resulting in progressive neurodegeneration that eventually leads to death.

By expressing the human Aβ protein in the fly, we have studied the mechanisms and toxicity of the aggregation in detail and how different cell types in the fly are affected. We have also used this model to investigate the effect of potential drugs that can have a positive impact on disease progression. In the first and second work in this thesis, we have, in a systematic way, proved that the length of the Aβ-peptide is essential for its toxicity and propensity to aggregate. If the peptide expressed ends at amino acid 42 it is extremely toxic to the fly nervous system. However, this toxicity can be completely abolished by expressing a variant that is shorter than 42 amino acids (1-37 to 1-41 aa), or be significantly reduced by expressing a longer variant (1-43 aa). Toxicity can be partly mitigated in trans by co-expressing the 1-42 variant with a 1-38 variant. This supports the theory that the disease progression could be inhibited if the formation of Aβ 1-42 is decreased. In the third work we demonstrate that amyloid aggregates can be found in various cell types of Drosophila, however, the toxicity seem to be selective to neurons. Our results indicate that the aggregates of glial expressing flies have a more mature structure, which appear to be less toxic. This also suggests that glial cells might spread Aβ aggregates without being harmed. The last work in this thesis investigates how curcumin (turmeric) can affect Aβ aggregation and toxicity. Curcumin appears to shift the equilibrium between the less stable

aggregates and mature fibers toward the final stage resulting in an improved lifespan for treated flies.

In summary, this thesis demonstrates that the toxicity of Aβ in Drosophila is highly dependent on the Aβ variant expressed, the structure of the protein aggregates and which cell type that expresses the protein. We have also shed light on the potential of using Drosophila when it comes to examining possible therapeutic substances as a tool for drug discovery.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2017. , p. 88
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1859
National Category
Chemical Sciences Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Cell and Molecular Biology Pharmacology and Toxicology
Identifiers
URN: urn:nbn:se:liu:diva-138389DOI: 10.3384/diss.diva-138389ISBN: 9789176855089 (print)OAI: oai:DiVA.org:liu-138389DiVA, id: diva2:1109702
Public defence
2017-06-09, Planck, Fysikhuset, Campus Valla, Linköping, 14:15 (English)
Opponent
Supervisors
Available from: 2017-06-14 Created: 2017-06-14 Last updated: 2018-04-25Bibliographically approved
List of papers
1. Systematic A beta Analysis in Drosophila Reveals High Toxicity for the 1-42, 3-42 and 11-42 Peptides, and Emphasizes N- and C-Terminal Residues
Open this publication in new window or tab >>Systematic A beta Analysis in Drosophila Reveals High Toxicity for the 1-42, 3-42 and 11-42 Peptides, and Emphasizes N- and C-Terminal Residues
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2015 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 7, article id e0133272Article in journal (Refereed) Published
Abstract [en]

Brain amyloid plaques are a hallmark of Alzheimers disease (AD), and primarily consist of aggregated A beta peptides. While A beta 1-40 and A beta 1-42 are the most abundant, a number of other A beta peptides have also been identified. Studies have indicated differential toxicity for these various A beta peptides, but in vivo toxicity has not been systematically tested. To address this issue, we generated improved transgenic Drosophila UAS strains expressing 11 pertinent A beta peptides. UAS transgenic flies were generated by identical chromosomal insertion, hence removing any transgenic position effects, and crossed to a novel and robust Gal4 driver line. Using this improved Gal4/UAS set-up, survival and activity assays revealed that A beta 1-42 severely shortens lifespan and reduces activity. N-terminal truncated peptides were quite toxic, with 3-42 similar to 1-42, while 11-42 showed a pronounced but less severe phenotype. N-terminal mutations in 3-42 (E3A) or 11-42 (E11A) resulted in reduced toxicity for 11-42, and reduced aggregation for both variants. Strikingly, C-terminal truncation of A beta (1-41, -40, -39, -38, -37) were non-toxic. In contrast, C-terminal extension to 1-43 resulted in reduced lifespan and activity, but not to the same extent as 1-42. Mutating residue 42 in 1-42 (A42D, A42R and A42W) greatly reduced A beta accumulation and toxicity. Histological and biochemical analysis revealed strong correlation between in vivo toxicity and brain A beta aggregate load, as well as amount of insoluble A beta. This systematic Drosophila in vivo and in vitro analysis reveals crucial N- and C-terminal specificity for A beta neurotoxicity and aggregation, and underscores the importance of residues 1-10 and E11, as well as a pivotal role of A42.

Place, publisher, year, edition, pages
Public Library of Science, 2015
National Category
Chemical Sciences Clinical Medicine
Identifiers
urn:nbn:se:liu:diva-120740 (URN)10.1371/journal.pone.0133272 (DOI)000358622000074 ()26208119 (PubMedID)
Note

Funding Agencies|Swedish VINNOVA; King Gustaf Vs and Queen Victorias Freemasons Foundation; AstraZeneca, Sodertalje; Swedish Research Council; VINNOVA grant, "Innovations for future health"

Available from: 2015-08-24 Created: 2015-08-24 Last updated: 2018-04-25
2. Curcumin Promotes A-beta Fibrillation and Reduces Neurotoxicity in Transgenic Drosophila
Open this publication in new window or tab >>Curcumin Promotes A-beta Fibrillation and Reduces Neurotoxicity in Transgenic Drosophila
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2012 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 2Article in journal (Refereed) Published
Abstract [en]

The pathology of Alzheimers disease (AD) is characterized by the presence of extracellular deposits of misfolded and aggregated amyloid-beta (A beta) peptide and intraneuronal accumulation of tangles comprised of hyperphosphorylated Tau protein. For several years, the natural compound curcumin has been proposed to be a candidate for enhanced clearance of toxic A beta amyloid. In this study we have studied the potency of feeding curcumin as a drug candidate to alleviate A beta toxicity in transgenic Drosophila. The longevity as well as the locomotor activity of five different AD model genotypes, measured relative to a control line, showed up to 75% improved lifespan and activity for curcumin fed flies. In contrast to the majority of studies of curcumin effects on amyloid we did not observe any decrease in the amount of A beta deposition following curcumin treatment. Conformation-dependent spectra from p-FTAA, a luminescent conjugated oligothiophene bound to A beta deposits in different Drosophila genotypes over time, indicated accelerated pre-fibrillar to fibril conversion of A beta(1-42) in curcumin treated flies. This finding was supported by in vitro fibrillation assays of recombinant A beta(1-42). Our study shows that curcumin promotes amyloid fibril conversion by reducing the pre-fibrillar/oligomeric species of A beta, resulting in a reduced neurotoxicity in Drosophila.

Place, publisher, year, edition, pages
Public Library of Science, 2012
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:liu:diva-73502 (URN)10.1371/journal.pone.0031424 (DOI)000302733900047 ()
Note
funding agencies|Knut and Alice Wallenberg foundation||Swedish Foundation for Strategic Research||Hjarnfonden||Swedish Research Council||Gustaf V. foundation||European Union||Available from: 2012-01-05 Created: 2012-01-05 Last updated: 2018-04-25

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