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α-Synuclein Fibril Formation and the Effects of Lipid Membranes

Gaspar, Ricardo LU (2018)
Abstract (Swedish)
Amyloid protein aggregation results in major disturbances of cellular processes in humans. The most common amyloid-related disorders are Alzheimer´s disease and Parkinson´s disease. Parkinson´s disease is charaterized by the formation of protein-rich aggregates that are deposited in neurons, termed Lewy bodies and Lewy neurites. The main component identified in these protein-rich deposits is the peptide α-synuclein.

The work described here is mainly focused on in vitro aggregation studies of α-synuclein alone and in the presence of lipid membranes of different compositions. This is possible as we have identified conditions leading to reproducible thioflavin-T aggregation kinetics. The aggregation mechanism of α-synuclein was... (More)
Amyloid protein aggregation results in major disturbances of cellular processes in humans. The most common amyloid-related disorders are Alzheimer´s disease and Parkinson´s disease. Parkinson´s disease is charaterized by the formation of protein-rich aggregates that are deposited in neurons, termed Lewy bodies and Lewy neurites. The main component identified in these protein-rich deposits is the peptide α-synuclein.

The work described here is mainly focused on in vitro aggregation studies of α-synuclein alone and in the presence of lipid membranes of different compositions. This is possible as we have identified conditions leading to reproducible thioflavin-T aggregation kinetics. The aggregation mechanism of α-synuclein was shown to be pH dependent. At neutral pH, both homogeneous primary nucleation and secondary processes are undetectable. At mildly acidic pH, present in the lumens of some intracellular compartments, secondary processes are enhanced. Experiments were performed to identify the nature of these secondary processes, distinguishing between fragmentation of fibrils and nucleation of monomers on the surface of existing aggregates. It was found that monomer-dependent secondary nucleation is the underlying dominant microscopic event of the aggregation process at mildly acidic pH, providing an autocatalytic amyloid amplification event. Crystallin chaperones purified from bovine eye lens were shown capable of inhibiting fibril formation of α-synuclein by affecting both secondary nucleation and elongation processes.

In amyloid plaques associated with several amyloidogenic diseases, tightly associated lipids have been identified. We explore how lipid membranes interfere with the aggregation mechanism of α-synuclein. Our results show a dependence of the aggregation rate on lipid composition and lipid charge. Biological exosomes, isolated from neuroblastoma cells, were shown to accelerate α-synuclein aggregation. This effect was found to be due to neuro-specific ganglioside lipids. Investigating the catalytic effect of anionic lipid membranes on α-synuclein aggregation, it was shown that charge-based interactions, induce conformational change of α-synuclein from random coil to α-helix, and is also associated with rapid aggregation kinetics. However, charge-independent interactions are also important, as lipid membranes containing uncharged lipid species with large hydrophilic headgroups were shown capable of triggering α-synuclein aggregation, although far less efficiently and without inducing α-synuclein conformational change to α-helix.

Investigating α-synuclein-ganglioside co-aggregation by imaging with cryo-EM at different time points along the aggregation reaction revealed aggregates with different structural morphology compared to those formed from pure α-synuclein. The images show a clear evolution of the vesicle size distribution, with a dramatic decrease in vesicle size during the lagphase of the aggregation reaction. Co-aggregates are thin and curly, decorated with small and monodisperse lipid vesicles contrary to compact bundles observed for fibrils formed from protein alone.
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Abstract
Amyloid protein aggregation results in major disturbances of cellular processes in humans. The most common amyloid-related disorders are Alzheimer´s disease and Parkinson´s disease. Parkinson´s disease is charaterized by the formation of protein-rich aggregates that are deposited in neurons, termed Lewy bodies and Lewy neurites. The main component identified in these protein-rich deposits is the peptide α-synuclein.

The work described here is mainly focused on in vitro aggregation studies of α-synuclein alone and in the presence of lipid membranes of different compositions. This is possible as we have identified conditions leading to reproducible thioflavin-T aggregation kinetics. The aggregation mechanism of α-synuclein was... (More)
Amyloid protein aggregation results in major disturbances of cellular processes in humans. The most common amyloid-related disorders are Alzheimer´s disease and Parkinson´s disease. Parkinson´s disease is charaterized by the formation of protein-rich aggregates that are deposited in neurons, termed Lewy bodies and Lewy neurites. The main component identified in these protein-rich deposits is the peptide α-synuclein.

The work described here is mainly focused on in vitro aggregation studies of α-synuclein alone and in the presence of lipid membranes of different compositions. This is possible as we have identified conditions leading to reproducible thioflavin-T aggregation kinetics. The aggregation mechanism of α-synuclein was shown to be pH dependent. At neutral pH, both homogeneous primary nucleation and secondary processes are undetectable. At mildly acidic pH, present in the lumens of some intracellular compartments, secondary processes are enhanced. Experiments were performed to identify the nature of these secondary processes, distinguishing between fragmentation of fibrils and nucleation of monomers on the surface of existing aggregates. It was found that monomer-dependent secondary nucleation is the underlying dominant microscopic event of the aggregation process at mildly acidic pH, providing an autocatalytic amyloid amplification event. Crystallin chaperones purified from bovine eye lens were shown capable of inhibiting fibril formation of α-synuclein by affecting both secondary nucleation and elongation processes.

In amyloid plaques associated with several amyloidogenic diseases, tightly associated lipids have been identified. We explore how lipid membranes interfere with the aggregation mechanism of α-synuclein. Our results show a dependence of the aggregation rate on lipid composition and lipid charge. Biological exosomes, isolated from neuroblastoma cells, were shown to accelerate α-synuclein aggregation. This effect was found to be due to neuro-specific ganglioside lipids. Investigating the catalytic effect of anionic lipid membranes on α-synuclein aggregation, it was shown that charge-based interactions, induce conformational change of α-synuclein from random coil to α-helix, and is also associated with rapid aggregation kinetics. However, charge-independent interactions are also important, as lipid membranes containing uncharged lipid species with large hydrophilic headgroups were shown capable of triggering α-synuclein aggregation, although far less efficiently and without inducing α-synuclein conformational change to α-helix.

Investigating α-synuclein-ganglioside co-aggregation by imaging with cryo-EM at different time points along the aggregation reaction revealed aggregates with different structural morphology compared to those formed from pure α-synuclein. The images show a clear evolution of the vesicle size distribution, with a dramatic decrease in vesicle size during the lagphase of the aggregation reaction. Co-aggregates are thin and curly, decorated with small and monodisperse lipid vesicles contrary to compact bundles observed for fibrils formed from protein alone.
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Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Riek, Roland, Swiss Federal Institute of Technology, (ETH), Zurich, Schweiz
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Protein misfolding, alpha-synuclein, Parkinson's disease, microscopic rate constants, Thioflavin T, ganglioside lipids, Exosomes, protein-lipid co-aggregation, model membranes, monomer-dependent secondary nucleation
pages
105 pages
publisher
Physical Chemistry, Lund University
defense location
Lecture Hall C, Kemicentrum, Naturvetarvägen 14, Lund
defense date
2018-10-26 09:00
ISBN
978-91-7422-599-0
978-91-7422-600-3
language
English
LU publication?
yes
id
6fa55de5-6121-4981-9715-3a995a381d93
date added to LUP
2018-09-27 16:00:28
date last changed
2018-11-21 21:41:52
@phdthesis{6fa55de5-6121-4981-9715-3a995a381d93,
  abstract     = {Amyloid protein aggregation results in major disturbances of cellular processes in humans. The most common amyloid-related disorders are Alzheimer´s disease and Parkinson´s disease. Parkinson´s disease is charaterized by the formation of protein-rich aggregates that are deposited in neurons, termed Lewy bodies and Lewy neurites. The main component identified in these protein-rich deposits is the peptide α-synuclein. <br/><br/>The work described here is mainly focused on in vitro aggregation studies of α-synuclein alone and in the presence of lipid membranes of different compositions. This is possible as we have identified conditions leading to reproducible thioflavin-T aggregation kinetics. The aggregation mechanism of α-synuclein was shown to be pH dependent. At neutral pH, both homogeneous primary nucleation and secondary processes are undetectable. At mildly acidic pH, present in the lumens of some intracellular compartments, secondary processes are enhanced. Experiments were performed to identify the nature of these secondary processes, distinguishing between fragmentation of fibrils and nucleation of monomers on the surface of existing aggregates. It was found that monomer-dependent secondary nucleation is the underlying dominant microscopic event of the aggregation process at mildly acidic pH, providing an autocatalytic amyloid amplification event. Crystallin chaperones purified from bovine eye lens were shown capable of inhibiting fibril formation of α-synuclein by affecting both secondary nucleation and elongation processes.<br/><br/>In amyloid plaques associated with several amyloidogenic diseases, tightly associated lipids have been identified. We explore how lipid membranes interfere with the aggregation mechanism of α-synuclein. Our results show a dependence of the aggregation rate on lipid composition and lipid charge. Biological exosomes, isolated from neuroblastoma cells, were shown to accelerate α-synuclein aggregation. This effect was found to be due to neuro-specific ganglioside lipids. Investigating the catalytic effect of anionic lipid membranes on α-synuclein aggregation, it was shown that charge-based interactions, induce conformational change of α-synuclein from random coil to α-helix, and is also associated with rapid aggregation kinetics. However, charge-independent interactions are also important, as lipid membranes containing uncharged lipid species with large hydrophilic headgroups were shown capable of triggering α-synuclein aggregation, although far less efficiently and without inducing α-synuclein conformational change to α-helix. <br/><br/>Investigating α-synuclein-ganglioside co-aggregation by imaging with cryo-EM at different time points along the aggregation reaction revealed aggregates with different structural morphology compared to those formed from pure α-synuclein. The images show a clear evolution of the vesicle size distribution, with a dramatic decrease in vesicle size during the lagphase of the aggregation reaction. Co-aggregates are thin and curly, decorated with small and monodisperse lipid vesicles contrary to compact bundles observed for fibrils formed from protein alone.<br/>},
  author       = {Gaspar, Ricardo},
  isbn         = {978-91-7422-599-0},
  keyword      = {Protein misfolding,alpha-synuclein,Parkinson's disease,microscopic rate constants,Thioflavin T,ganglioside lipids,Exosomes,protein-lipid co-aggregation,model membranes,monomer-dependent secondary nucleation},
  language     = {eng},
  pages        = {105},
  publisher    = {Physical Chemistry, Lund University},
  school       = {Lund University},
  title        = {α-Synuclein Fibril Formation and the Effects of Lipid Membranes},
  year         = {2018},
}