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In situ proximity ligation assay reveals co-localization of alpha-synuclein and SNARE proteins in murine primary neurons
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap, Geriatrik.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap, Geriatrik.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap, Geriatrik.
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Medicinska fakulteten, Institutionen för folkhälso- och vårdvetenskap, Geriatrik.ORCID-id: 0000-0001-5466-8370
Vise andre og tillknytning
2018 (engelsk)Inngår i: Frontiers in Neurology, ISSN 1664-2295, E-ISSN 1664-2295, Vol. 9, artikkel-id 180Artikkel i tidsskrift (Fagfellevurdert) 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

sted, utgiver, år, opplag, sider
Frontiers Media S.A., 2018. Vol. 9, artikkel-id 180
HSV kategori
Identifikatorer
URN: urn:nbn:se:uu:diva-342580DOI: 10.3389/fneur.2018.00180ISI: 000428063500001PubMedID: 29623065OAI: oai:DiVA.org:uu-342580DiVA, id: diva2:1184870
Forskningsfinansiär
Marianne and Marcus Wallenberg FoundationThe Swedish Brain FoundationSwedish Society of MedicineMagnus Bergvall FoundationTilgjengelig fra: 2018-02-22 Laget: 2018-02-22 Sist oppdatert: 2018-06-28bibliografisk kontrollert
Inngår i avhandling
1. Characterization of Physiological and Pathological Alpha-Synuclein: Implications for Parkinson’s Disease and Related Disorders
Åpne denne publikasjonen i ny fane eller vindu >>Characterization of Physiological and Pathological Alpha-Synuclein: Implications for Parkinson’s Disease and Related Disorders
2018 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2018. s. 65
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1434
Emneord
Alpha-synuclein, Aggregation, Parkinson’s disease, Dementia with Lewy bodies, Oxidative stress, Antibodies, Synapses, SNARE
HSV kategori
Forskningsprogram
Molekylär medicin
Identifikatorer
urn:nbn:se:uu:diva-342761 (URN)978-91-513-0248-5 (ISBN)
Disputas
2018-04-13, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjöldsväg 20, Uppsala, 09:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2018-03-21 Laget: 2018-02-23 Sist oppdatert: 2018-04-24

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