Structural and functional aspects of membranes : : the involvement of lipid rafts in Alzheimer's disease pathogenesis : the interplay between protein oligomers and plasma membrane physicochemical features in determining cytotoxicity / / Elisa Evangelisti.

• Superior document: Premio Tesi di dottorato
• VerfasserIn: Evangelisti, Elisa,
• Place / Publishing House: Firenze : : Firenze University Press,, 2013.
• Year of Publication: 2013
• Language: English
• Series: Premio Tesi di dottorato.
• Physical Description: 1 online resource (147 pages) :; illustrations.

Alzheimer's disease (AD) is a common form of dementia characterized by the formation of extracellular senile plaques composed of aggregated amyloid peptide (Aβ). The present studies provide evidence that: cell resistance to amyloid toxicity is related to lipid raft cholesterol content. Choleste...

Table of Contents

• Summary
• Introduction
• 1.1 Protein aggregation and amyloidoses
• 1.1.1 Mechanisms of amyloid fibril formation
• 1.1.2 Oligomer structural polymorphism: fibrillar and prefibrillar oligomers
• 1.1.3 Oligomer toxicity: common mechanism of pathogenesis
• 1.1.4 Amyloid formation is an inherent property of polypeptide chains: functional amyloid and disease unreleated amyloid
• 1.2 HypF-N: model protein of amyloid aggregation unrelated to disease
• 1.2.1 Function, structure and aggregation of HypF-N
• 1.2.2 HypF-N protofibrils interact with cell membranes originating a cytotoxic cascade
• 1.2.3 A causative link between the structure of HypF-N oligomers and their ability to cause cellular dysfunction
• 1.3 Alzheimer's disease
• 1.3.1 The Alzheimer phenotype
• 1.3.2 The elaborate processing of APP
• 1.3.3 The genetics of Alzheimer's disease
• 1.3.4 Peripheral cells as a tool to identify and test hypotheses on AD pathophysiology
• 1.3.5 Adult neurogenesis and stem cell technology for AD
• 1.4 Cholesterol and gangliosides in the central nervous system (CNS)
• 1.4.1 Brain cholesterol metabolism
• 1.4.2 Ganglioside metabolism
• 1.4.3 Lipid rafts
• 1.4.4 Role of cholesterol in AD
• 1.4.5 Role of gangliosides in AD
• 1.5 Aim of the study
• Materials & Methods
• 2.1 Materials
• 2.1.1 Chemicals
• 2.1.2 Fluorescent probes
• 2.1.3 Peptides and aggregation protocols
• 2.2 Cell cultures
• 2.3 Methods
• 2.3.1 Separation processes
• 2.3.2 Differentiation of human mesenchymal stromal cells
• 2.3.3 Modulation of membrane cholesterol levels
• 2.3.4 Modulation of membrane GM1 levels
• 2.3.5 Cholesterol content measurements
• 2.3.6 GM1 content measurements
• 2.3.7 Cell exposure to peptide aggregates
• 2.3.8 Analysis of aggregate interaction with the cells
• 2.3.9 Analysis of aggregate interaction with GM1
• 2.3.10 Analysis of aggregate internalisation
• 2.3.11 Analysis of membrane permeability
• 2.3.12 Analysis of cytosolic Ca2+ dyshomeostasis
• 2.3.13 Evaluation of ROS production
• 2.3.14 Analysis of lipid peroxidation
• 2.3.15 Cytotoxicity assay and cell death analysis: apoptotic and necrotic markers
• 2.3.16 Steady-state fluorescence anisotropy
• 2.3.17 Atomic force microscopy (AFM)
• 2.3.18 Measurements of the fluorescence intensities
• 2.3.19 Statistical analysis
• Results
• 3.1 Results I
• 3.1.1 A protective role for lipid raft cholesterol against amyloidinduced membrane damage in human neuroblastoma cells
• 3.1.2 Aß42 oligomer binding to the cell surface and its cytotoxic effect are modulated by membrane cholesterol content
• 3.1.3 Aß42 oligomers colocalize with lipid rafts
• 3.1.4 Isolation and characterization of DRMs
• 3.1.5 Effects of ADDLs on lipid raft structural order
• 3.1.6 AFM imaging of supported DRMs purified from cells exposed to ADDLs
• 3.2 Results II
• 3.2.1 Lipid rafts mediate amyloid-induced calcium dyshomeostasis and oxidative stress in Alzheimer's disease
• 3.2.2 Lipid rafts are primary interaction sites for Aß42 oligomers at the plasma membrane
• 3.2.3 Cholesterol and GM1 mediate Aß42 accumulation at the plasma membrane
• 3.2.4 Cholesterol and GM1 mediate Ca2+ dyshomeostasis and extensive membrane permeabilization induced by Aß42 oligomers
• 3.2.5 GM1 modulates lipid peroxidation and cytotoxicity induced by Aß42 oligomers
• 3.2.6 GM1 mediates Aß42-induced Ca2+ dyshomeostasis, lipid peroxidation and cytotoxicity in rat cortical neurons
• 3.3 Results III
• 3.3.1 Membrane lipid composition and its physicochemical properties define cell vulnerability to aberrant protein oligomers
• 3.3.2 Membrane cholesterol content modulates oligomer cytotoxicity
• 3.3.3 Membrane cholesterol modulates oligomer-induced alteration of intracellular Ca2+ homeostasis and ROS levels
• 3.3.4 Cholesterol levels modulate membrane permeability to the oligomers
• 3.3.5 Membrane GM1 affects the cytotoxic and permeabilizing effects of HypF-N oligomers
• 3.3.6 GM1, rather than cholesterol, plays a dominant role in oligomer cytotoxicity and membrane permeability
• 3.4 Results IV
• 3.4.1 Neuronal differentiation of human mesenchymal stromal cells increases their resistance to Aß42 aggregate toxicity
• 3.4.2 Neuronal differentiation of hMSCs results in reduced levels of membrane GM1
• 3.4.3 Neuronal differentiation of hMSCs reduces the interaction of Aß42 oligomers with the cell surface
• 3.4.4 Neuronal differentiation of hMSCs reduces Aß42 oligomerinduced intracellular Ca2+ dyshomeostasis and oxidative stress
• 3.4.5 Neuronal differentiation of hMSCs increases cell resistance to Aß42 aggregates Discussion
• 4.1 A protective role for lipid raft cholesterol against amyloid-induced membrane damage in human neuroblastoma cells
• 4.2 Lipid rafts mediate amyloid-induced calcium dyshomeostasis and membrane permeabilization in Alzheimer's fibroblasts
• 4.3 Membrane lipid composition and its physicochemical properties define cell vulnerability to aberrant protein oligomers
• 4.4 Neuronal differentiation of human mesenchymal stromal cells increases their resistance to Aß42 aggregate toxicity
• 4.5 Concluding remarks
• Abbreviations
• References.