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Characterization of Physiological and Pathological Alpha-Synuclein: Implications for Parkinson’s Disease and Related Disorders
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences. (Molecular geriatrics)
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Aggregated alpha-synuclein is the main component of Lewy bodies and Lewy neurites, intraneuronal inclusions found in the brains of Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) patients (synucleinopathies). Alpha-synuclein is a presynaptic protein, which is most commonly an unfolded monomer in its physiological state. However, under pathological conditions it can start to misfold and enter an aggregation pathway that will lead to the formation of oligomers of increasing size and finally insoluble fibrils. The oligomers have been hypothesized to be the most neurotoxic species, but studies of their properties have been hindered by their heterogeneity and kinetic instability. The overall aim of this thesis was to characterize and compare physiological and pathological forms of alpha-synuclein from different sources: recombinant monomers, oligomers formed in vitro through exposure to oxidative stress related reactive aldehydes, aggregates from a synucleinopathy mouse model and from synucleinopathy patients.

In paper I we studied the effect of low molar excess of two lipid peroxidation products, 4-oxo-2-nonenal (ONE) and 4-hydroxy-2-nonenal (HNE), on the oligomerization of alpha-synuclein. Through biophysical methods we observed that, although both aldehydes bound to alpha-synuclein directly, ONE produced SDS-stable oligomers more rapidly than HNE. Moreover, ONE induced oligomerization at both acidic and neutral pH, while HNE only formed oligomers at neutral pH.

In paper II we mapped the surface exposed epitopes of in vitro and in vivo generated alpha-synuclein species by using immunoglobulin Y antibodies raised against short linear peptides covering most of the alpha-synuclein sequence. Monomers were found to react with most antibodies, while the latter part of the N-terminus and mid-region of HNE oligomers and fibrils was found to be occluded in oligomers and fibrils. Through immunohistochemistry we compared alpha-synuclein aggregates in brain tissue from patients with synucleinopathies as well as from a mouse model expressing A30P human alpha-synuclein. Although the exposed epitopes were found to be similar overall, subtle differences were detected in the C-terminus.

An additional aim of this thesis was to characterize synaptic aggregates of alpha-synuclein. In paper III we obtained synaptosomal preparations of the A30P mouse model and found that a subset of the alpha-synuclein present in the synaptosomes was proteinase K resistant and therefore aggregated. Further biochemical analyses showed that the aggregated alpha-synuclein mainly was of human, i.e. transgenic, origin and that Ser 129 was not phosphorylated, which otherwise is a common post translational modification of alpha-synuclein in Lewy bodies.

It has been suggested that alpha-synuclein plays a role in neurotransmitter release by binding to the SNARE protein VAMP-2 and thereby chaperoning the SNARE complex assembly. In paper IV we used proximity ligation assay to visualize the co-localization of alpha-synuclein and the SNARE proteins in primary neurons from non-transgenic and A30P transgenic mice.

In conclusion, in this thesis we have characterized a variety of alpha-synuclein species and shed light on the diversity of alpha-synuclein aggregates. Additionally, we have characterized synaptic species of alpha-synuclein and analyzed the co-localization between alpha-synuclein and SNARE proteins in neurons.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. , p. 65
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1434
Keywords [en]
Alpha-synuclein, Aggregation, Parkinson’s disease, Dementia with Lewy bodies, Oxidative stress, Antibodies, Synapses, SNARE
National Category
Neurosciences Biochemistry and Molecular Biology
Research subject
Molecular Medicine
Identifiers
URN: urn:nbn:se:uu:diva-342761ISBN: 978-91-513-0248-5 (print)OAI: oai:DiVA.org:uu-342761DiVA, id: diva2:1185119
Public defence
2018-04-13, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjöldsväg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2018-03-21 Created: 2018-02-23 Last updated: 2018-04-24
List of papers
1. Low molar excess of 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote oligomerization of alpha-synuclein through different pathways
Open this publication in new window or tab >>Low molar excess of 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote oligomerization of alpha-synuclein through different pathways
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2017 (English)In: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 110, p. 421-431Article in journal (Refereed) Published
Abstract [en]

Aggregated alpha-synuclein is the main component of Lewy bodies, intraneuronal inclusions found in brains with Parkinson's disease and dementia with Lewy bodies. A body of evidence implicates oxidative stress in the pathogenesis of these diseases. For example, a large excess (30:1, aldehyde:protein) of the lipid peroxidation end products 4-oxo-2-nonenal (ONE) or 4-hydroxy-2-nonenal (HNE) can induce alpha-synuclein oligomer formation. The objective of the study was to investigate the effect of these reactive aldehydes on alpha-synuclein at a lower molar excess (3:1) at both physiological (7.4) and acidic (5.4) pH. As observed by size-exclusion chromatography, ONE rapidly induced the formation of alpha-synuclein oligomers at both pH values, but the effect was less pronounced under the acidic condition. In contrast, only a small proportion of alpha-synuclein oligomers were formed with low excess HNE-treatment at physiological pH and no oligomers at all under the acidic condition. With prolonged incubation times (up to 96 h), more alpha-synuclein was oligomerized at physiological pH for both ONE and HNE. As determined by Western blot, ONE-oligomers were more SDS-stable and to a higher-degree cross-linked as compared to the HNE-induced oligomers. However, as shown by their greater sensitivity to proteinase K treatment, ONE-oligomers, exhibited a less compact structure than HNE-oligomers. As indicated by mass spectrometry, ONE modified most Lys residues, whereas HNE primarily modified the His50 residue and fewer Lys residues, albeit to a higher degree than ONE. Taken together, our data show that the aldehydes ONE and HNE can modify alpha-synuclein and induce oligomerization, even at low molar excess, but to a higher degree at physiological pH and seemingly through different pathways.

Keywords
Alpha-synuclein, Oligomers, 4-oxo-2-nonenal, 4-hydroxy-2-nonenal, Oxidative stress
National Category
Medical and Health Sciences Engineering and Technology Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-326663 (URN)10.1016/j.freeradbiomed.2017.07.004 (DOI)000406049200038 ()28690195 (PubMedID)
Funder
Swedish Research Council, 2011-4519, 2012-2172, 2010-6745Marianne and Marcus Wallenberg FoundationThe Swedish Brain FoundationSwedish Society of MedicineÅke Wiberg Foundation
Note

Correction in: Free Radical Biology and Medicine, vol. 117, pages 258-258.

DOI: 10.1016/j.freeradbiomed.2018.02.007

Available from: 2017-07-19 Created: 2017-07-19 Last updated: 2019-04-29Bibliographically approved
2. Mapping of Surface-Exposed Epitopes of In Vitro and In Vivo Aggregated Species of Alpha-Synuclein
Open this publication in new window or tab >>Mapping of Surface-Exposed Epitopes of In Vitro and In Vivo Aggregated Species of Alpha-Synuclein
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2017 (English)In: Cellular and molecular neurobiology, ISSN 0272-4340, E-ISSN 1573-6830, Vol. 37, no 7, p. 1217-1226Article in journal (Refereed) Published
Abstract [en]

Aggregated alpha-synuclein is the main component of Lewy bodies, intraneuronal deposits observed in Parkinson's disease and dementia with Lewy bodies. The objective of the study was to identify surface-exposed epitopes of alpha-synuclein in vitro and in vivo formed aggregates. Polyclonal immunoglobulin Y antibodies were raised against short linear peptides of the alpha-synuclein molecule. An epitope in the N-terminal region (1-10) and all C-terminal epitopes (90-140) were found to be exposed in an indirect enzyme-linked immunosorbent assay (ELISA) using recombinant monomeric, oligomeric, and fibrillar alpha-synuclein. In a phospholipid ELISA, the N-terminus and mid-region of alpha-synuclein (i.e., 1-90) were associated with phosphatidylserine and thus occluded from antibody binding. The antibodies that reacted most strongly with epitopes in the in vitro aggregates (i.e., 1-10 and epitopes between positions 90-140) also labeled alpha-synuclein inclusions in brains from transgenic (Thy-1)-h[A30P] alpha-synuclein mice and Lewy bodies and Lewy neurites in brains of patients with alpha-synucleinopathies. However, differences in reactivity were observed with the C-terminal antibodies when brain tissue from human and transgenic mice was compared. Taken together, the study shows that although similar epitopes are exposed in both in vitro and in vivo formed alpha-synuclein inclusions, structural heterogeneity can be observed between different molecular species.

Keywords
Parkinson's disease, Dementia with Lewy bodies, Alpha-synuclein, Epitope mapping
National Category
Geriatrics
Identifiers
urn:nbn:se:uu:diva-334384 (URN)10.1007/s10571-016-0454-0 (DOI)000409352200007 ()28028735 (PubMedID)
Funder
Swedish Research Council, 2011-4519Marianne and Marcus Wallenberg FoundationThe Swedish Brain FoundationSwedish Society of Medicine
Available from: 2017-12-15 Created: 2017-12-15 Last updated: 2020-02-04Bibliographically approved
3. Characterization of synaptic aggregates of alpha-synuclein in (Thy-1)-h[A30P] alpha-synuclein mice
Open this publication in new window or tab >>Characterization of synaptic aggregates of alpha-synuclein in (Thy-1)-h[A30P] alpha-synuclein mice
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(English)Manuscript (preprint) (Other academic)
National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-342341 (URN)
Available from: 2018-02-20 Created: 2018-02-20 Last updated: 2018-02-23
4. In situ proximity ligation assay reveals co-localization of alpha-synuclein and SNARE proteins in murine primary neurons
Open this publication in new window or tab >>In situ proximity ligation assay reveals co-localization of alpha-synuclein and SNARE proteins in murine primary neurons
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2018 (English)In: Frontiers in Neurology, ISSN 1664-2295, E-ISSN 1664-2295, Vol. 9, article id 180Article in journal (Refereed) Published
Abstract [en]

The aggregation of alpha-synuclein (alpha Syn) is the pathological hallmark of Parkinson's disease, dementia with Lewy bodies and related neurological disorders. However, the physiological function of the protein and how this function relates to its pathological effects remain poorly understood. One of the proposed roles of aSyn is to promote the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly by binding to VAMP-2. The objective of this study was to visualize the co-localization between aSyn and the SNARE proteins (VAMP-2, SNAP-25, and syntaxin-1) for the first time using in situ proximity ligation assay (PLA). Cortical primary neurons were cultured from either non-transgenic or transgenic mice expressing human aSyn with the A30P mutation under the Thy-1 promoter. With an antibody recognizing both mouse and human aSyn, a PLA signal indicating close proximity between aSyn and the three SNARE proteins was observed both in the soma and throughout the processes. No differences in the extent of PLA signals were seen between non-transgenic and transgenic neurons. With an antibody specific against human aSyn, the PLA signal was mostly located to the soma and was only present in a few cells. Taken together, in situ PLA is a method that can be used to investigate the co-localization of aSyn and the SNARE proteins in primary neuronal cultures

Place, publisher, year, edition, pages
Frontiers Media S.A., 2018
National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-342580 (URN)10.3389/fneur.2018.00180 (DOI)000428063500001 ()29623065 (PubMedID)
Funder
Marianne and Marcus Wallenberg FoundationThe Swedish Brain FoundationSwedish Society of MedicineMagnus Bergvall Foundation
Available from: 2018-02-22 Created: 2018-02-22 Last updated: 2018-06-28Bibliographically approved

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