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. Cholesterol and GM1, affect the susceptibility of Familial Alzheimer's Disease (FAD) broblasts to Aβ42 oligomers in opposite ways, by modulating amyloid binding to lipid rafts and its subsequent toxic effects. The degree of toxicity of the oligomeric species results from a complex interplay between the structural and physicochemical features of both the oligomers and the cellular membrane. Neuronal differentiation of human mesenchymal stromal cells increases their resistance to Aβ42 aggregate toxicity.

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.