Insights into the function and dysfunction of α-synuclein in cells

C.C. Raiss

Research output: ThesisPhD Thesis - Research UT, graduation UTAcademic

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Abstract

This thesis sheds light on the function and dysfunction of the protein α-synuclein (α-S) in the test tube and in cells and ultimately its possible involvement in Parkinson’s disease (PD). Following the introduction in Chapter 1, Chapters 2 and 3 concentrate on the investigation of the interaction between the proteins α-S and actin. In Chapter 2 we determine their dissociation constants with microscale thermophoresis and reveal that α-S has a high affinity for G-actin. The determined interaction strength was even comparable to other G-actin binding proteins. Despite this high binding affinity and the postulated function of α-S as a G-actin regulating protein, the binding of α-S to G-actin has no effect on the actin polymerization kinetics. Instead, α-S seems to bind to and stabilize F-actin structures. Chapter 3 reports on how the formation of G-actin/αS complexes attenuates the lag time of α-S aggregation depending on the actin concentration. Whereas low actin concentrations inhibit the onset of α-S aggregation this effect disappears at higher concentrations. However, although the presence of actin can inhibit the onset of aggregation it cannot prevent it. Chapter 4 draws attention to the intra-cellular occurrence of α-S spots, smaller than the diffraction limit, consisting of many proteins. To study and visualize these diffraction-limited α-S spots, we made use of stimulated emission depletion (STED) microscopy. The colocalization of these α-S clusters with a membrane marker and the appearance of α-S on the surface of giant intracellular vesicles suggest that that the spots are small vesicles and α-S functions as membrane binding protein. Chapter 5 focuses on cell models in which α-S inclusion formation is induced in cells. We describe the resemblance of induced α-S inclusion to LBs. Moreover, we not only observe different morphologies, but also observe that α-S aggregates even occur in functionally different inclusions both in vitro and in vivo. Finally, in chapter 6, we investigate the effect of exogenous α-S on the viability and electrophysiology of neuronal networks. Whereas the viability was unaffected, we noted development of α-S aggregates in cells and a progressive decrease in neuronal connectivity and excitability.
Original languageEnglish
Awarding Institution
  • University of Twente
Supervisors/Advisors
  • Claessens, Mireille M.A.E., Supervisor
  • Subramaniam, Vinod, Supervisor
Award date2 Apr 2015
Place of PublicationEnschede, the Netherlands
Publisher
Print ISBNs978-94-6108-945-8
Publication statusPublished - 2 Apr 2015

Fingerprint

Synucleins
Actins
Protein S
Microfilament Proteins
Electrophysiology
Protein Binding
Polymerization
Parkinson Disease
Microscopy

Keywords

  • IR-95450
  • METIS-310104

Cite this

Raiss, C. C. (2015). Insights into the function and dysfunction of α-synuclein in cells. Enschede, the Netherlands: University of Twente.
Raiss, C.C.. / Insights into the function and dysfunction of α-synuclein in cells. Enschede, the Netherlands : University of Twente, 2015. 148 p.
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Raiss, CC 2015, 'Insights into the function and dysfunction of α-synuclein in cells', University of Twente, Enschede, the Netherlands.

Insights into the function and dysfunction of α-synuclein in cells. / Raiss, C.C.

Enschede, the Netherlands : University of Twente, 2015. 148 p.

Research output: ThesisPhD Thesis - Research UT, graduation UTAcademic

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AU - Raiss, C.C.

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AB - This thesis sheds light on the function and dysfunction of the protein α-synuclein (α-S) in the test tube and in cells and ultimately its possible involvement in Parkinson’s disease (PD). Following the introduction in Chapter 1, Chapters 2 and 3 concentrate on the investigation of the interaction between the proteins α-S and actin. In Chapter 2 we determine their dissociation constants with microscale thermophoresis and reveal that α-S has a high affinity for G-actin. The determined interaction strength was even comparable to other G-actin binding proteins. Despite this high binding affinity and the postulated function of α-S as a G-actin regulating protein, the binding of α-S to G-actin has no effect on the actin polymerization kinetics. Instead, α-S seems to bind to and stabilize F-actin structures. Chapter 3 reports on how the formation of G-actin/αS complexes attenuates the lag time of α-S aggregation depending on the actin concentration. Whereas low actin concentrations inhibit the onset of α-S aggregation this effect disappears at higher concentrations. However, although the presence of actin can inhibit the onset of aggregation it cannot prevent it. Chapter 4 draws attention to the intra-cellular occurrence of α-S spots, smaller than the diffraction limit, consisting of many proteins. To study and visualize these diffraction-limited α-S spots, we made use of stimulated emission depletion (STED) microscopy. The colocalization of these α-S clusters with a membrane marker and the appearance of α-S on the surface of giant intracellular vesicles suggest that that the spots are small vesicles and α-S functions as membrane binding protein. Chapter 5 focuses on cell models in which α-S inclusion formation is induced in cells. We describe the resemblance of induced α-S inclusion to LBs. Moreover, we not only observe different morphologies, but also observe that α-S aggregates even occur in functionally different inclusions both in vitro and in vivo. Finally, in chapter 6, we investigate the effect of exogenous α-S on the viability and electrophysiology of neuronal networks. Whereas the viability was unaffected, we noted development of α-S aggregates in cells and a progressive decrease in neuronal connectivity and excitability.

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KW - METIS-310104

M3 - PhD Thesis - Research UT, graduation UT

SN - 978-94-6108-945-8

PB - University of Twente

CY - Enschede, the Netherlands

ER -

Raiss CC. Insights into the function and dysfunction of α-synuclein in cells. Enschede, the Netherlands: University of Twente, 2015. 148 p.