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Characterization of α-synuclein oligomers: Implications for Lewy Body Disorders
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences. (Lannfelt)
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy are disorders featuring accumulation of Lewy bodies in brain. The main component of these large insoluble intracellular inclusions is the presynaptic protein alpha-synuclein (α-synuclein). It is generally believed that α-synuclein monomers adopt an abnormal conformation that favors the formation of soluble oligomers or protofibrils and, eventually, insoluble fibrils depositing as Lewy bodies. Notably, the intermediately sized oligomers/protofibrils seem to have particular neurotoxic effects. Several factors may influence the formation of α-synuclein oligomers/protofibrils, e.g. the reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) formed during oxidative stress.

The overall aims of this thesis were to investigate biophysical and biochemical properties of in vitro generated α-synuclein oligomers, characterize their functional effects on cell and animal disease models as well as to explore whether their formation could be prevented in a cell culture model for oligomerization. 

Here, it was found that α-synuclein rapidly formed oligomers after incubation with both ONE and HNE. The resulting oligomers were stable and did not continue to form insoluble fibrils. By comparing HNE- and ONE induced α-synuclein oligomers biochemically they were both found to exhibit extensive β-beta sheet structure and had a molecular size of ~2000 kDa. However, they differed in morphology; the ONE induced α-synuclein oligomers described round amorphous species whereas the HNE induced α-synuclein oligomers appeared as elongated protofibril-like structures. Both these oligomers were cell internalized to varying degrees and induced toxicity in neuroblastoma cells.

In addition, the ONE induced α-synuclein oligomers seemed to initiate aggregation of monomeric α-synuclein in vitro, but failed to do so in vivo.

Finally, treatment of α-synuclein overexpressing cells with monoclonal antibodies specific for α-synuclein significantly reduced aggregation and lowered levels of the protein, suggesting increased turnover in these cells. 

To conclude, this thesis has characterized different oligomeric α-synuclein species, which may have properties similar to soluble species central to the pathogenesis of Parkinson’s disease and other disorders with α-synuclein pathology. For therapeutic strategies it is important to selectively target such harmful protein species and avoid interaction with other forms of α-synuclein, which may have vital physiological cellular functions.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2011. , 70 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 717
Keyword [en]
Parkinson’s disease; Alpha-synuclein; Lewy bodies; Oligomers; Reactive aldehydes; monoclonal antibody
National Category
Neurosciences Geriatrics
Research subject
Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-160102ISBN: 978-91-554-8198-8 (print)OAI: oai:DiVA.org:uu-160102DiVA: diva2:450418
Public defence
2011-12-02, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjöldsväg 20, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2011-11-11 Created: 2011-10-16 Last updated: 2011-11-23Bibliographically approved
List of papers
1. The lipid peroxidation metabolite 4-oxo-2-nonenal cross-links alpha-synuclein causing rapid formation of stable oligomers
Open this publication in new window or tab >>The lipid peroxidation metabolite 4-oxo-2-nonenal cross-links alpha-synuclein causing rapid formation of stable oligomers
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2009 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 378, no 4, 872-876 p.Article in journal (Refereed) Published
Abstract [en]

Recently, the aldehyde 4-oxo-2-nonenal (ONE) was identified as a product of lipid peroxidation and found to be an effective protein modifier. In this in vitro study we investigated structural implications of the interaction between ONE and alpha-synuclein, a protein which forms intraneuronal inclusions in neurodegenerative disorders such as Parkinson's disease and dementia with Lewy bodies. Our results demonstrate that ONE induced an almost complete conversion of monomeric alpha-synuclein into 40-80 nm wide and 6-8 nm high soluble beta-sheet-rich oligomers with a molecular weight of approximately 2000 kDa. Furthermore, the ONE-induced alpha-synuclein oligomers displayed a high stability and were not sensitive to treatment with sodium dodecyl sulfate, indicating that ONE stabilized the oligomers by cross-linking individual alpha-synuclein molecules. Despite prolonged incubation the oligomers did not continue to aggregate into a fibrillar state, thus suggesting that these alpha-synuclein species were not on a fibrillogenic pathway.

Keyword
α-Synuclein, 4-Oxo-2-nonenal, Oligomers, Parkinson’s disease, Dementia with Lewy bodies, Oxidative stress
National Category
Medical and Health Sciences Engineering and Technology
Identifiers
urn:nbn:se:uu:diva-111550 (URN)10.1016/j.bbrc.2008.12.005 (DOI)000262447400036 ()19070597 (PubMedID)
Available from: 2009-12-16 Created: 2009-12-16 Last updated: 2017-12-12Bibliographically approved
2. The lipid peroxidation products 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote the formation of α-synuclein oligomers with distinct biochemical, morphological, and functional properties
Open this publication in new window or tab >>The lipid peroxidation products 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote the formation of α-synuclein oligomers with distinct biochemical, morphological, and functional properties
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2011 (English)In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 50, no 3, 428-437 p.Article in journal (Refereed) Published
Abstract [en]

Oxidative stress has been implicated in the etiology of neurodegenerative disorders with alpha-synuclein pathology. Lipid peroxidation products such as 4-oxo-2-nonenal (ONE) and 4-hydroxy-2-nonenal (HNE) can covalently modify and structurally alter proteins. Herein, we have characterized ONE- or HNE-induced alpha-synuclein oligomers. Our results demonstrate that both oligomers are rich in beta-sheet structure and have a molecular weight of about 2000 kDa. Atomic force microscopy analysis revealed that ONE-induced alpha-synuclein oligomers were relatively amorphous, with a diameter of 40-80 nm and a height of 4-8 nm. In contrast, the HNE-induced alpha-synuclein oligomers had a protofibril-like morphology with a width of 100-200 nm and a height of 2-4 nm. Furthermore, neither oligomer type polymerized into amyloid-like fibrils despite prolonged incubation. Although more SDS and urea stable, because of a higher degree of cross-linking, ONE-induced alpha-synuclein oligomers were less compact and more sensitive to proteinase K treatment. Finally, both ONE- and HNE-induced alpha-synuclein oligomers were cytotoxic when added exogenously to a neuroblastoma cell line, but HNE-induced alpha-synuclein oligomers were taken up by the cells to a significantly higher degree. Despite nearly identical chemical structures, ONE and HNE induce the formation of off-pathway alpha-synuclein oligomers with distinct biochemical, morphological, and functional properties.

Keyword
alpha-Synuclein, Oligomers, Lewy bodies, Lipid peroxidation, 4-Oxo-2-nonenal, Oxidative stress, 4-Hydroxy-2-nonenal, Free radicals
National Category
Medical and Health Sciences Materials Engineering
Research subject
Engineering Science with specialization in Microsystems Technology
Identifiers
urn:nbn:se:uu:diva-148623 (URN)10.1016/j.freeradbiomed.2010.11.027 (DOI)000287429600003 ()21130160 (PubMedID)
Available from: 2011-03-08 Created: 2011-03-08 Last updated: 2017-12-11Bibliographically approved
3. Off-pathway alpha-synuclein oligomers seem to alter alpha-synuclein turnover in a cell model but lack seeding capability in vivo
Open this publication in new window or tab >>Off-pathway alpha-synuclein oligomers seem to alter alpha-synuclein turnover in a cell model but lack seeding capability in vivo
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2013 (English)In: Amyloid: Journal of Protein Folding Disorders, ISSN 1350-6129, E-ISSN 1744-2818, Vol. 20, no 4, 233-244 p.Article in journal (Refereed) Published
Abstract [en]

Aggregated α-synuclein is the major component of Lewy bodies, protein inclusions observed in the brain in neurodegenerative disorders such as Parkinson’s disease and dementia with Lewy bodies. Experimental evidence indicates that α-synuclein potentially can be transferred between cells and act as a seed to accelerate the aggregation process. Here, we investigated in vitro and in vivo seeding effects of α-synuclein oligomers induced by the reactive aldehyde 4-oxo-2-nonenal (ONE). As measured by a Thioflavin-T based fibrillization assay, there was an earlier onset of aggregation when α-synuclein oligomers were added to monomeric α-synuclein. In contrast, exogenously added α-synuclein oligomers did not induce aggregation in a cell model. However, cells overexpressing α-synuclein that were treated with the oligomers displayed reduced α-synuclein levels, indicating that internalized oligomers either decreased the expression or accelerated the degradation of transfected α-synuclein. Also in vivo there were no clear seeding effects, as intracerebral injections of α-synuclein oligomers into the neocortex of α-synuclein transgenic mice did not induce formation of Proteinase K resistant α-synuclein pathology. Taken together, we could observe a seeding effect of the ONE-induced α-synuclein oligomers in a fibrillization assay, but neither in a cell nor in a mouse model.

Keyword
Aggregation, alpha-synuclein, oligomers, Parkinson’s disease, seeding
National Category
Cell and Molecular Biology Neurosciences Engineering and Technology
Research subject
Geriatrics; Neuroscience; Engineering Science with specialization in Microsystems Technology
Identifiers
urn:nbn:se:uu:diva-160100 (URN)10.3109/13506129.2013.835726 (DOI)000327304800006 ()
Available from: 2011-10-20 Created: 2011-10-16 Last updated: 2017-12-08
4. Antibodies against alpha-synuclein reduce oligomerization in living cells
Open this publication in new window or tab >>Antibodies against alpha-synuclein reduce oligomerization in living cells
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2011 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 10, e27230- p.Article in journal (Refereed) Published
Abstract [en]

Recent research implicates soluble aggregated forms of α-synuclein as neurotoxic species with a central role in the pathogenesis of Parkinson’s disease and related disorders. The pathway by which α-synuclein aggregates is believed to follow a step-wise pattern, in which dimers and smaller oligomers are initially formed. Here, we used H4 neuroglioma cells expressing α-synuclein fused to hemi:GFP to study the effects of α-synuclein monoclonal antibodies on the early stages of aggregation, as quantified by Bimolecular Fluorescence Complementation assay. Widefield and confocal microscopy revealed that cells treated for 48 h with monoclonal antibodies internalized antibodies to various degrees. Oligomer-selective and C-terminal specific α-synuclein antibodies reduced the extent of α-synuclein dimerization/oligomerization, as indicated by decreased GFP fluorescence signal. Furthermore, ELISA measurements on lysates and conditioned media from antibody treated cells displayed lower α-synuclein levels compared to untreated cells, suggesting increased protein turnover. Taken together, our results propose that extracellular administration of monoclonal antibodies can modify or inhibit early steps in the aggregation process of α-synuclein, thus providing further support for passive immunization against diseases with α-synuclein pathology.

Keyword
Alpha-synuclein; Parkinson’s disease; Lewy bodies; Aggregation; Bimolecular Fluorescence Complementation; Monoclonal antibodies; Immunotherapy
National Category
Neurosciences Cell and Molecular Biology
Research subject
Neuroscience; Geriatrics
Identifiers
urn:nbn:se:uu:diva-160097 (URN)10.1371/journal.pone.0027230 (DOI)000296916000059 ()22073131 (PubMedID)
Available from: 2011-10-20 Created: 2011-10-16 Last updated: 2017-12-08Bibliographically approved

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