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Understanding the dual nature of lysozyme: part villain – part hero: A Drosophila melanogaster model of lysozyme amyloidosis
Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Amyloid proteins are a distinct class of proteins that can misfold into β-sheet rich structures that later mature to form the characteristic species known as amyloid fibrils, and accumulate in tissues in the human body. The misfolding event is often caused by mutations (or outer factors such as changes in pH) that destabilize the native protein structure. The mature amyloid fibrils were initially believed to be associated with diseases connected to protein misfolding such as Alzheimer’s disease (AD), Parkinson’s disease, transthyretin amyloidosis and lysozyme amyloidosis. However, now it is known that many different factors are involved in these diseases such as failure in protein clearance, lysosomal dysfunction and formation of intermediate misfolded protein species, which possess cytotoxic properties, preceding the formation of mature fibrils.

In this thesis the amyloidogenic protein lysozyme has been examined in vivo by using Drosophila melanogaster (fruit fly) as a model organism. The effects of over-expressing human lysozyme and amyloidogenic variants in Drosophila have been investigated both in the absence and presence of the serum amyloid P component (SAP), a protein known to interact with amyloid species. In addition, the role of lysozyme in AD has been investigated by  co-expressing human lysozyme and amyloid β in Drosophila.

The lysozyme protein is an enzyme naturally found in bodily fluids such as tears, breast milk and saliva. It is engaged in the body’s defense and acts by hydrolyzing the cell wall of invading bacteria. Certain disease-associated point mutations in the gene encoding lysozyme destabilize the protein and cause it to misfold which results in systemic amyloidosis. To investigate the in vivo misfolding behavior of lysozyme we developed and established a Drosophila model of lysozyme amyloidosis. SAP is commonly found attached to amyloid deposits in the body; however, the role of SAP in amyloid diseases is unknown. To investigate the effect of SAP in lysozyme misfolding, these two proteins were co-expressed in Drosophila.

The amyloid β peptide is involved in AD, building up the plaques found in AD patient brains. These plaques trigger neuroinflammation and since lysozyme is upregulated during various inflammation conditions, a possible role of lysozyme in AD was investigated by overexpressing lysozyme in a Drosophila model of AD. Interaction between lysozyme and the amyloid β protein was also studied by biophysical measurements.

During my work with this thesis, the dual nature of lysozyme emerged; on the one hand a villain, twisted by mutations, causing the lysozyme amyloidosis disease. On the other hand a hero, delaying the toxicity and maybe the neurological damage caused by the amyloid β peptide.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2014. , 67 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1574
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-106647DOI: 10.3384/diss.diva-106647ISBN: 978-91-7519-405-9 (print)OAI: oai:DiVA.org:liu-106647DiVA: diva2:717677
Public defence
2014-05-05, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 10:15 (English)
Opponent
Supervisors
Available from: 2014-05-16 Created: 2014-05-16 Last updated: 2014-05-19Bibliographically approved
List of papers
1. Disease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster
Open this publication in new window or tab >>Disease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster
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2012 (English)In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 26, no 1, 192-202 p.Article in journal (Refereed) Published
Abstract [en]

We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic variants F57I and D67H were expressed in Drosophila melanogaster using the UAS-gal4 system and both the ubiquitous and retinal expression drivers Act5C-gal4 and gmr-gal4. The nontransgenic w(1118) Drosophila line was used as a control throughout. We utilized ELISA experiments to probe lysozyme protein levels, scanning electron microscopy for eye phenotype classification, and immunohistochemistry to detect the unfolded protein response (UPR) activation. We observed that expressing the destabilized F57I and D67H lysozymes triggers UPR activation, resulting in degradation of these variants, whereas the WT lysozyme is secreted into the fly hemolymph. Indeed, the level of WT was up to 17 times more abundant than the variant proteins. In addition, the F57I variant gave rise to a significant disruption of the eye development, and this correlated to pronounced UPR activation. These results support the concept that the onset of familial amyloid disease is linked to an inability of the UPR to degrade completely the amyloidogenic lysozymes prior to secretion, resulting in secretion of these destabilized variants, thereby leading to deposition and associated organ damage.-Kumita, J. R., Helmfors, L., Williams, J., Luheshi, L. M., Menzer, L., Dumoulin, M., Lomas, D. A., Crowther, D. C., Dobson, C. M., Brorsson, A.-C. Disease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster.

Place, publisher, year, edition, pages
Federation of American Society of Experimental Biology (FASEB), 2012
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-73186 (URN)10.1096/fj.11-185983 (DOI)000299202200020 ()21965601 (PubMedID)
Note

funding agencies|Swedish Research Council||Biotechnology and Biological Sciences Research Council| BB/E019927/1 BBH0038431 |Wellcome Trust||Leverhulme Trust||European Commission| LSHM-CT-2006-037525/EURAMY |Medical Research Council| G0700990 |VINNOVA||

Available from: 2011-12-21 Created: 2011-12-21 Last updated: 2017-12-08
2. SAP to the rescue: Serum amyloid p component ameliorates neurological damage caused by expressing a lysozyme variant in the central nervous system of Drosophila melanogaster
Open this publication in new window or tab >>SAP to the rescue: Serum amyloid p component ameliorates neurological damage caused by expressing a lysozyme variant in the central nervous system of Drosophila melanogaster
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Lysozyme amyloidosis is a hereditary disease in which mutations in the gene encoding lysozyme leads to misfolding and consequently accumulation of amyloid material. To improve understanding of the processes involved we expressed human wild type (WT) lysozyme and the disease-associated variant F57I in the central nervous system (CNS) of a Drosophila melanogaster model of lysozyme amyloidosis, with and without serum amyloid p component (SAP). We found that flies expressing the amyloidogenic variant F57I in the CNS have a shorter lifespan and lower locomotor activity than flies expressing WT lysozyme or control flies, indicating that the flies’ neurological functions are impaired when F57I is expressed in the nerve cells. In addition, the Unfolded Protein Response (UPR) was upregulated in the F57I-expressing flies. However, co-expression of SAP in the CNS restored the F57I flies’ locomotor activity and lifespan. Thus, SAP has apparent ability to protect nerve cells from damage caused by F57I. Furthermore, co-expression of SAP prevented accumulation of insoluble forms of lysozyme in both WT- and F57I-expressing flies and delayed up-regulation of the UPR by 10 days in F57I flies. Our findings suggest that SAP can prevent cytotoxic effects of expressing F57I in fly CNS by retaining F57I in a soluble form and preventing crowding of misfolded F57I species in the endoplasmic reticulum.

Keyword
xbp1-EGFP, ER stress, iFly, Amyloidosis, Misfolding
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-106644 (URN)
Available from: 2014-05-16 Created: 2014-05-16 Last updated: 2014-05-16
3. Co-expression of a disease-associated lysozyme variant with human lysozyme in Drosophila causes accumulation of amyloid deposits and neurodegeneration
Open this publication in new window or tab >>Co-expression of a disease-associated lysozyme variant with human lysozyme in Drosophila causes accumulation of amyloid deposits and neurodegeneration
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Lysozyme amyloidosis is a dominantly inherited form of amyloid disease. Mutant variants of the protein, with increased tendencies to aggregate compared to the wild type (WT), accumulate in large amyloid deposits in multiple organs, eventually leading to organ failure. Humans affected by lysozyme amyloidosis carry one allele for the wild type protein and one allele encoding for a mutant variant of lysozyme. We have used a Drosophila melanogaster model to investigate the effect of co-expressing WT lysozyme and a mutated variant, F57I, in the central nervous system (CNS) of the fly. In this study, using activity and longevity assays, WT-F57I flies showed a lower activity and a shorter lifespan than flies expressing only WT or the F57I variant of lysozyme (median survival 16 days compared to 34 and 23 respectively). This indicates deteriorating neurological functions in WT-F57I flies; exceeding the decrease in neurological function previously observed for flies only expressing the mutated variant, F57I. In addition, accumulation of insoluble species with amyloid structure was detected for the WT-F57I flies but not for the WT or the F57I flies. Our study show that co-expression of WT lysozyme and the amyloidogenic variant F57I results in neurological damage and is required for accumulation of amyloid deposits, which is characteristic for the disease observed in humans. Our data suggest that insoluble amyloid species or intermediate species, formed on the pathway toward amyloid species, may be cytotoxic and thus contribute to the impaired neurological functions observed for the WT-F57I flies.

National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-106645 (URN)
Available from: 2014-05-16 Created: 2014-05-16 Last updated: 2014-05-16Bibliographically approved
4. A protective role of lysozyme in Alzheimer disease
Open this publication in new window or tab >>A protective role of lysozyme in Alzheimer disease
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Alzheimer disease (AD) is a devastating neurodegenerative disorder where extracellular plaques composed of amyloid β (Aβ) peptides and neuroinflammation are some of the main hallmarks of the disease. Activated microglial cells, which are the resident macrophages in the central nervous system, are suggested to trigger the inflammation response in AD. To discover neuroinflammation biomarkers would be important to reveal the pathological mechanisms of AD and develop therapies that target inflammation mediators. Lysozyme is part of the innate immune system and is secreted from macrophages during various inflammation conditions. However, the involvement of lysozyme in AD pathology has not been explored previously. We have discovered that lysozyme is up-regulated in cerebrospinal fluid from AD patients. Cells exposed to Aβ increased the expression of lysozyme indicating that Aβ might be responsible for the upregulation of lysozyme detected in cerebrospinal fluid. In vitro studies revealed that lysozyme binds to monomeric Aβ1-42 and alters the aggregation pathway counteracting formation of toxic Aβ species. In a newly developed Drosophila model, co-expression of lysozyme with Aβ in brain neurons reduced the formation of insoluble Aβ species, prolonged the survival and improved the activity of the double transgenic flies compared to flies only expressing Aβ. Our findings identify lysozyme as a modulator of Aβ aggregation and toxicity and our discoveries has the potential to be used for development of new treatment strategies and to use lysozyme as a biomarker for AD.

Keyword
Lysozyme, Biomarker, Alzheimer disease, Drosophila, CSF, Aβ aggregation
National Category
Natural Sciences
Identifiers
urn:nbn:se:liu:diva-106646 (URN)
Available from: 2014-05-16 Created: 2014-05-16 Last updated: 2014-05-16Bibliographically approved

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