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Cellular Uptake of alpha-Synuclein Oligomer-Selective Antibodies is Enhanced by the Extracellular Presence of alpha-Synuclein and Mediated via Fc gamma Receptors
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Geriatrics.
BioArctic Neurosci AB, Stockholm, Sweden..
BioArctic Neurosci AB, Stockholm, Sweden..
Univ Med Ctr Gottingen, Dept Neurodegenerat & Restorat Res, Gottingen, Germany..
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2017 (English)In: Cellular and molecular neurobiology, ISSN 0272-4340, E-ISSN 1573-6830, Vol. 37, no 1, 121-131 p.Article in journal (Refereed) Published
Abstract [en]

Immunotherapy targeting aggregated alpha-synuclein has emerged as a potential treatment strategy against Parkinson's disease and other alpha-synucleinopathies. We have developed alpha-synuclein oligomer/protofibril selective antibodies that reduce toxic alpha-synuclein in a human cell line and, upon intraperitoneal administration, in spinal cord of transgenic mice. Here, we investigated under which conditions and by which mechanisms such antibodies can be internalized by cells. For this purpose, human neuroglioma H4 cells were treated with either monoclonal oligomer/protofibril selective alpha-synuclein antibodies, linear epitope monoclonal alpha-synuclein antibodies, or with a control antibody. The oligomer/protofibril selective antibody mAb47 displayed the highest cellular uptake and was therefore chosen for additional analyses. Next, alpha-synuclein overexpressing cells were incubated with mAb47, which resulted in increased antibody internalization as compared to non-transfected cells. Similarly, regular cells exposed to mAb47 together with media containing alpha-synuclein displayed a higher uptake as compared to cells incubated with regular media. Finally, different Fc gamma receptors were targeted and we then found that blockage of Fc gamma RI and Fc gamma RIIB/C resulted in reduced antibody internalization. Our data thus indicate that the robust uptake of the oligomer/protofibril selective antibody mAb47 by human CNS-derived cells is enhanced by extracellular alpha-synuclein and mediated via Fc gamma receptors. Altogether, our finding lend further support to the belief that alpha-synuclein pathology can be modified by monoclonal antibodies and that these can target toxic alpha-synuclein species in the extracellular milieu. In the context of immunotherapy, antibody binding of alpha-synuclein would then not only block further aggregation but also mediate internalization and subsequent degradation of antigen-antibody complexes.

Place, publisher, year, edition, pages
2017. Vol. 37, no 1, 121-131 p.
Keyword [en]
alpha-Synuclein, Parkinson's disease, Lewy bodies, Monoclonal antibodies, Antibody uptake, Fc gamma receptors
National Category
Geriatrics Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-316048DOI: 10.1007/s10571-016-0352-5ISI: 000392407200013PubMedID: 26961542OAI: oai:DiVA.org:uu-316048DiVA: diva2:1076817
Funder
Swedish Research Council, 2011-4519 2012-2172 2010-6745The Swedish Brain FoundationThe Dementia Association - The National Association for the Rights of the DementedMagnus Bergvall FoundationGerman Research Foundation (DFG)
Available from: 2017-02-24 Created: 2017-02-24 Last updated: 2017-11-29Bibliographically approved
In thesis
1. Alpha-Synuclein Oligomers: Cellular Mechanisms and Aspects of Antibody Treatment
Open this publication in new window or tab >>Alpha-Synuclein Oligomers: Cellular Mechanisms and Aspects of Antibody Treatment
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In Parkinson’s disease (PD) and dementia with Lewy bodies (DLB), aggregated α-synuclein deposit inside cells within the brain. Smaller soluble α-synuclein aggregates, oligomers, are present both intra- and extracellularly. The α-synuclein oligomers are known to be particularly harmful, although the underlying neurotoxic mechanisms are not fully understood. The aim of this thesis was to investigate the pathogenic roles of α-synuclein oligomers and the possibility to target such species with antibody treatment.

Passive immunotherapy with α-synuclein antibodies can lead to reduced pathology and ameliorated symptoms in transgenic mice. However, it remains unknown whether the antibodies are taken up by cells or whether they act extracellularly. In Paper I, we assessed cellular internalization of various α-synuclein monoclonal antibodies. The oligomer selective mAb47 displayed the highest uptake, which was promoted by the extracellular presence of α-synuclein.

Alpha-synuclein aggregates can be found in both neurons and glial cells, but the pathogenic role of glial deposits has only been sparsely investigated. In Paper II, co-cultures of neurons and glia were exposed to α-synuclein oligomers. The astrocytes in the cultures rapidly accumulated oligomers, which were only partially degraded by lysosomes. The sustained intracellular α-synuclein deposits were associated with mitochondrial stress reactions in the astrocytes. 

In Paper III, we sought to explore whether the astrocytic pathology induced by α-synuclein oligomers could be ameliorated by antibody treatment. Pre-incubation of oligomers with mAb47 promoted α-synuclein clearance, reduced astrocytic accumulation and rescued cells from mitochondrial stress. We could demonstrate that binding of the antibody to its antigen in the extracellular space was crucial for these effects to occur.

The progressive pathology in PD is believed to be driven by cell-to-cell spreading of α-synuclein aggregates, potentially via exosomes and other extracellular vesicles (EVs). In Paper IV, we found that either fusing α-synuclein to a non-physiological protein tag or introducing the PD-causing A53T mutation directed α-synuclein towards EV secretion. Also, EV-associated α-synuclein was particularly prone to induce toxicity in recipient cells.

In conclusion, this thesis sheds new light on the cellular dysfunction related to α-synuclein pathology and on how the underlying pathogenic processes may be targeted by antibody treatment.  

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. 64 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1344
Keyword
Parkinson's Disease, Alpha-synuclein, Aggregation, Oligomers, Monoclonal Antibody, Glia, Astrocyte, Immunofluorescence
National Category
Other Medical Sciences not elsewhere specified
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-326320 (URN)978-91-513-0008-5 (ISBN)
Public defence
2017-09-14, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2017-08-23 Created: 2017-07-05 Last updated: 2017-09-08

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Gustafsson, GabrielLannfelt, LarsBergström, JoakimIngelsson, Martin

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