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Studies of α-synuclein Oligomers-with Relevance to Lewy Body Disorders
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences. (Lannfelt)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The protein alpha-synuclein (α-synuclein) accumulates in the brain in disorders such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). It is believed that the monomeric form of α-synuclein can adopt a partially folded structure and start to aggregate and form intermediately sized oligomers or protofibrils. The aggregation process can continue with the formation of insoluble fibrils, which are deposited as Lewy bodies. The oligomers/protofibrils have been shown to be toxic to neurons and are therefore believed to be involved in the pathogenesis of the actual diseases.      

The overall aims of this thesis were to investigate the properties of α-synuclein oligomers and to generate and characterize antibodies against these species. In addition, the potential for immunotherapy of the α-synuclein oligomer-selective antibodies were evaluated in a transgenic mouse model with α-synuclein pathology.

Stable, β-sheet rich α-synuclein oligomers were induced by incubation with either one of the reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). The oligomers exhibited distinct morphological properties, although both types were toxic when added to a neuroblastoma cell line. The seeding effects of ONE-induced oligomers were studied in vitro and in vivo. The oligomers induced seeding of monomeric α-synuclein in a fibrillization assay but not in a cell model or when injected intracerebrally in transgenic mice. It seemed, however, as if the oligomers affected α-synuclein turnover in the cell model.

By immunizing mice with HNE-induced oligomers antibody producing hybridomas were generated. Three monoclonal antibodies were found to have strong selectivity for α-synuclein oligomers. These antibodies recognized Lewy body pathology in brains from patients with PD and DLB as well as inclusions in the brain from young α-synuclein transgenic mice, but did not bind to other amyloidogenic proteins. Finally, immunotherapy with one of the oligomer/protofibril selective antibodies resulted in lower levels of such α-synuclein species in the spinal cord of α-synuclein transgenic mice.

To conclude, this thesis has focused on characterizing properties of α-synuclein oligomers. In particular, antibodies selectively targeting such neurotoxic forms were generated and evaluated for passive immunization in a transgenic mouse model. Such immunotherapy may represent a future treatment strategy against Lewy body disorders.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2013. , 85 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 925
Keyword [en]
Alpha-synuclein, Parkinson´s disease, Lewy body dementia, Oligomer, Monoclonal antibody, Immunotherapy, Reactive aldehydes
National Category
Neurosciences
Research subject
Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-204466ISBN: 978-91-554-8720-1 (print)OAI: oai:DiVA.org:uu-204466DiVA: diva2:640048
Public defence
2013-09-27, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2013-09-06 Created: 2013-08-06 Last updated: 2013-09-06
List of papers
1. 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
2. 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
3. Monoclonal antibodies selective for α-synuclein oligomers/protofibrils recognize brain pathology in Lewy body disorders and α-synuclein transgenic mice with the disease-causing A30P mutation
Open this publication in new window or tab >>Monoclonal antibodies selective for α-synuclein oligomers/protofibrils recognize brain pathology in Lewy body disorders and α-synuclein transgenic mice with the disease-causing A30P mutation
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2013 (English)In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 126, no 1, 131-144 p.Article in journal (Refereed) Published
Abstract [en]

Inclusions of intraneuronal alpha-synuclein (-synuclein) can be detected in brains of patients with Parkinson's disease and dementia with Lewy bodies. The aggregation of -synuclein is a central feature of the disease pathogenesis. Among the different -synuclein species, large oligomers/protofibrils have particular neurotoxic properties and should therefore be suitable as both therapeutic and diagnostic targets. Two monoclonal antibodies, mAb38F and mAb38E2, with high affinity and strong selectivity for large -synuclein oligomers were generated. These antibodies, which do not bind amyloid-beta or tau, recognize Lewy body pathology in brains from patients with Parkinson's disease and dementia with Lewy bodies and detect pathology earlier in -synuclein transgenic mice than linear epitope antibodies. An oligomer-selective sandwich ELISA, based on mAb38F, was set up to analyze brain extracts of the transgenic mice. The overall levels of -synuclein oligomers/protofibrils were found to increase with age in these mice, although the levels displayed a large interindividual variation. Upon subcellular fractionation, higher levels of -synuclein oligomers/protofibrils could be detected in the endoplasmic reticulum around the age when behavioral disturbances develop. In summary, our novel oligomer-selective -synuclein antibodies recognize relevant pathology and should be important tools to further explore the pathogenic mechanisms in Lewy body disorders. Moreover, they could be potential candidates both for immunotherapy and as reagents in an assay to assess a potential disease biomarker.

Keyword
alpha-synuclein, dementia with Lewy bodies, monoclonal antibody, oligomer, Parkinson's disease, protofibril
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-204098 (URN)10.1111/jnc.12175 (DOI)000320722700014 ()
Available from: 2013-07-23 Created: 2013-07-22 Last updated: 2017-12-06Bibliographically approved
4. Immunotherapy targeting α-synuclein protofibrils reduced pathology in (Thy-1)-h[A30P] α-synuclein mice
Open this publication in new window or tab >>Immunotherapy targeting α-synuclein protofibrils reduced pathology in (Thy-1)-h[A30P] α-synuclein mice
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2014 (English)In: Neurobiology of Disease, ISSN 0969-9961, E-ISSN 1095-953X, Vol. 69, 134-143 p.Article in journal (Refereed) Published
Abstract [en]

Several lines of evidence suggest that accumulation of aggregated alpha-synuclein (α-synuclein) in the central nervous system (CNS) is an early pathogenic event and therefore a suitable therapeutic target in Parkinson’s disease and other Lewy body disorders. In recent years, animal studies have indicated immunotherapy with antibodies directed against α-synuclein as a promising novel treatment strategy. Since large α-synuclein oligomers, or protofibrils, have been demonstrated to possess pronounced cytotoxic properties, such species should be particularly attractive as therapeutic targets. An α-synuclein protofibril-selective monoclonal antibody, mAb47, was evaluated in the (Thy-1)-h[A30P] α-synuclein transgenic mouse model, featuring an age- and motor dysfunction-associated increase of α-synuclein protofibrils in the CNS. As measured by ELISA, mAb47-treated mice displayed significantly lower levels of both soluble and membrane-associated protofibrils in the spinal cord. In addition, a trend for increased survival as a result of reduced motor symptoms was observed with antibody treatment. Taken together, this study demonstrates reduced levels of pathogenic α-synuclein and indicates a reduction of motor dysfunction in transgenic mice upon peripheral administration of an α-synuclein protofibril-selective antibody. Thus, immunotherapy with antibodies targeting toxic α-synuclein species holds promise as a future disease-modifying treatment in Parkinson’s disease and related disorders.

Keyword
Alpha-synuclein, Parkinson´s disease, Oligomers, Monoclonal antibody, Immunotherapy
National Category
Immunology in the medical area Neurosciences
Research subject
Neuroscience; Geriatrics
Identifiers
urn:nbn:se:uu:diva-204463 (URN)10.1016/j.nbd.2014.05.009 (DOI)000339538900013 ()24851801 (PubMedID)
Note

Manuscript title: Reduced pathology in (Thy-1)-h [A30P]α-synuclein transgenic mice following immunotherapy with an α-synuclein protofibril-selective antibody

Alternative title: Immunotherapy targeting alpha-synuclein protofibrils reduced pathology in (Thy-1)-h[A30P]alpha-synuclein mice

Available from: 2013-08-09 Created: 2013-08-06 Last updated: 2017-12-06Bibliographically approved

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