Elisa Evangelisti

A causative link between the structure of aberrant protein oligomers and their toxicity

The aberrant assembly of peptides and proteins into fibrillar aggregates proceeds through oligomeric intermediates that are thought to be the primary pathogenic species in many protein deposition diseases. We describe two types of oligomers formed by the HypF-N protein that are morphologically and tinctorially similar, as detected with atomic force microscopy and thioflavin T assays, though one is benign when added to cell cultures whereas the other is toxic. Structural investigation at a residue-specific level using site-directed labeling with pyrene indicated differences in the packing of the hydrophobic interactions between adjacent protein molecules in the oligomers. The lower degree of hydrophobic packing was found to correlate with a higher ability to penetrate the cell membrane and cause an influx of Ca(2+) ions. Our findings suggest that structural flexibility and hydrophobic exposure are primary determinants of the ability of oligomeric assemblies to cause cellular dysfunction and its consequences, such as neurodegeneration.

A protective role for lipid raft cholesterol against amyloid-induced membrane damage in human neuroblastoma cells

Increasing evidence supports the idea that the initial events of Abeta oligomerization and cytotoxicity in Alzheimers disease involve the interaction of amyloid Abeta-derived diffusible ligands (ADDLs) with the cell membrane. This also indicates lipid rafts, ordered membrane microdomains enriched in cholesterol, sphingolipids and gangliosides, as likely primary interaction sites of ADDLs. To shed further light on the relation between ADDL-cell membrane interaction and oligomer cytotoxicity, we investigated the dependence of ADDLs binding to lipid rafts on membrane cholesterol content in human SH-SY5Y neuroblastoma cells. Confocal laser microscopy showed that Abeta1-42 oligomers markedly interact with membrane rafts and that a moderate enrichment of membrane cholesterol prevents their association with the monosialoganglioside GM1. Moreover, anisotropy fluorescence measurements of flotillin-1-positive rafts purified by sucrose density gradient suggested that the content of membrane cholesterol and membrane perturbation by ADDLs are inversely correlated. Finally, contact mode atomic force microscope images of lipid rafts in liquid showed that ADDLs induce changes in raft morphology with the appearance of large cavities whose size and depth were significantly reduced in similarly treated cholesterol-enriched rafts. Our data suggest that cholesterol reduces amyloid-induced membrane modifications at the lipid raft level by altering raft physicochemical features.

Membrane lipid composition and its physicochemical properties define cell vulnerability to aberrant protein oligomers

Increasing evidence suggests that the interaction of misfolded protein oligomers with cell membranes is a primary event resulting in the cytotoxicity associated with many protein-misfolding diseases, including neurodegenerative disorders. We describe here the results of a study on the relative contributions to toxicity of the physicochemical properties of protein oligomers and the cell membrane with which they interact. We altered the amount of cholesterol and the ganglioside GM1 in membranes of SH-SY5Y cells. We then exposed the cells to two types of oligomers of the prokaryotic protein HypF-N with different ultrastructural and cytotoxicity properties, and to oligomers formed by the amyloid-β peptide associated with Alzheimers disease. We identified that the degree of toxicity of the oligomeric species is the result of a complex interplay between the structural and physicochemical features of both the oligomers and the cell membrane.

A comparison of the biochemical modifications caused by toxic and non-toxic protein oligomers in cells

Peptides and proteins can convert from their soluble forms into highly ordered fibrillar aggregates, giving rise to pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. It is increasingly recognized that protein oligomers forming early in the process of fibril aggregation represent the pathogenic species in protein deposition diseases. The N‐terminal domain of the HypF protein from Escherichia coli (HypF‐N) has previously been shown to form, under distinct conditions, two types of HypF‐N oligomers with indistinguishable morphologies but distinct structural features at the molecular level. Only the oligomer type exposing hydrophobic surfaces and possessing sufficient structural plasticity is toxic (type A), whereas the other type is benign to cultured cells (type B). Here we show that only type A oligomers are able to induce a Ca2+ influx from the cell medium to the cytosol, to penetrate the plasma membrane, to increase intracellular reactive oxygen species production, lipid peroxidation and release of intracellular calcein, resulting in the activation of the apoptotic pathway. Remarkably, these oligomers can also induce a loss of cholinergic neurons when injected into rat brains. By contrast, markers of cellular stress and viability were unaffected in cultured and rat neuronal cells exposed to type B oligomers. The analysis of the time scales of such effects indicates that the difference of toxicity between the two oligomer types involve the early events of the toxicity cascade, shedding new light on the mechanism of action of protein oligomers and on the molecular targets for the therapeutic intervention against protein deposition diseases.

Novel S-acyl glutathione derivatives prevent amyloid oxidative stress and cholinergic dysfunction in Alzheimer disease models

Oxidative stress-mediated neuronal death may be initiated by a decrease in glutathione (GSH), whose levels are reduced in mitochondrial and synaptosomal fractions of specific CNS regions in Alzheimer disease (AD) patients. Currently, the use of GSH as a therapeutic agent is limited by its unfavorable pharmacokinetic properties. In this study, we designed the synthesis of new S-acyl glutathione (acyl-SG) thioesters of fatty acids via N-acyl benzotriazole-intermediate production and investigated their potential for targeted delivery of the parent GSH and free fatty acid to amyloid-exposed fibroblasts from familial AD patients and human SH-SY5Y neuroblastoma cells. Cell culture supplementation with acyl-SG derivatives triggers a significant decrease in lipid peroxidation and mitochondrial dysfunction in a fatty acid unsaturation degree-dependent fashion. Acyl-SG thioesters also protect cholinergic neurons against Aβ-induced damage and reduce glial reaction in rat brains. Collectively, these findings suggest that acyl-SG thioesters could prove useful as a tool for controlling AD-induced cerebral deterioration.

Lipid rafts are primary mediators of amyloid oxidative attack on plasma membrane

Increasing evidence indicates that cell surfaces are early interaction sites for Aβ-derived diffusible ligands (ADDLs) and neurons in Alzheimer’s disease (AD) pathogenesis. Our previous data showed significant oxidative damage at the plasma membrane in fibroblasts from familial AD patients with enhanced Aβ production. Here, we report that lipid rafts, ordered membrane microdomains, are chronic mediators of Aβ-induced lipid peroxidation in SH-SY5Y human neuroblastoma cells overexpressing amyloid precursor protein (APPwt) and APPV717G genes and in fibroblasts bearing the APPV717I gene mutation. Confocal microscope analysis showed that Aβ-oxidised rafts recruit more ADDLs than corresponding domains in control cells, triggering a further increase in membrane lipid peroxidation and loss of membrane integrity. Moreover, amyloid pickup at the oxidative-damaged domains was prevented by enhanced cholesterol levels, anti-ganglioside (GM1) antibodies, the B subunit of cholera toxin and lipid raft structure alteration. An enhanced structural rigidity of the detergent-resistant domains, isolated from APPwt and APPV717G cells and exposed to ADDLs, indicates a specific perturbation of raft physicochemical features in cells facing increased amyloid assembly at the membrane surface. These data identify lipid rafts as key mediators of oxidative damage as a result of their ability to recruit aggregates to the cell surface.

Membrane cholesterol enrichment prevents Aβ-induced oxidative stress in Alzheimer's fibroblasts

A growing body of evidence implicates low membrane cholesterol in the pathogenesis of Alzheimers disease (AD). Here we show that Aβ42 soluble oligomers accumulate more slowly and in reduced amount at the plasma membranes of PS-1L392V and APPV717I fibroblasts from familial AD (FAD) patients enriched in cholesterol content than at the counterpart membranes. The Aβ42-induced production of reactive oxygen species (ROS) and the increase in membrane lipoperoxidation were also prevented by high membrane cholesterol, thus resulting in a higher resistance to amyloid toxicity with respect to control fibroblasts. On the other hand, the recruitment of amyloid assemblies to the plasma membrane of cholesterol-depleted fibroblasts was significantly increased, thus triggering an earlier and sharper production of ROS and a higher membrane oxidative injury. These results identify membrane cholesterol as being key to Aβ42 oligomer accumulation at the cell surfaces and to the following Aβ42-induced cell death in AD neurons.

Extracellular chaperones prevent Aβ42-induced toxicity in rat brains.

Alzheimers disease (AD) is a progressive neurodegenerative disorder characterised by cognitive decline, formation of the extracellular amyloid β (Aβ42) plaques, neuronal and synapse loss, and activated microglia and astrocytes. Extracellular chaperones, which are known to inhibit amyloid fibril formation and promote clearance of misfolded aggregates, have recently been shown to reduce efficiently the toxicity of HypF-N misfolded oligomers to immortalised cell lines, by binding and clustering them into large species. However, the role of extracellular chaperones on Aβ oligomer toxicity remains unclear, with reports often appearing contradictory. In this study we microinjected into the hippocampus of rat brains Aβ42 oligomers pre-incubated for 1h with two extracellular chaperones, namely clusterin and α2-macroglobulin. The chaperones were found to prevent Aβ42-induced learning and memory impairments, as assessed by the Morris Water Maze test, and reduce Aβ42-induced glia inflammation and neuronal degeneration in rat brains, as probed by fluorescent immunohistochemical analyses. Moreover, the chaperones were able to prevent Aβ42 colocalisation with PSD-95 at post-synaptic terminals of rat primary neurons, suppressing oligomer cytotoxicity. All such effects were not effective by adding pre-formed oligomers and chaperones without preincubation. Molecular chaperones have therefore the potential to prevent the early symptoms of AD, not just by inhibiting Aβ42 aggregation, as previously demonstrated, but also by suppressing the toxicity of Aβ42 oligomers after they are formed. These findings elect them as novel neuroprotectors against amyloid-induced injury and excellent candidates for the design of therapeutic strategies against AD.

Lipid Rafts Mediate Amyloid-Induced Calcium Dyshomeostasis and Oxidative Stress in Alzheimer’s Disease

Several lines of evidence suggest that the initial events of amyloid-β peptide (Aβ) oligomerization and deposition in Alzheimers disease (AD) involve the interaction of soluble oligomers with neuronal membranes. In this study, we show that Aβ42 oligomers are recruited to lipid rafts, which are ordered membrane microdomains rich in cholesterol and gangliosides, resulting in lipid peroxidation, Ca(2+) dyshomeostasis and membrane permeabilization in primary fibroblasts from familial AD patients (FAD) bearing APPVal717Ile, PS-1Leu392Val or PS-1Met146Leu gene mutations. Moreover, the presence of significantly higher levels of lipid peroxidation correlated with greater structural modification in detergent resistant domains (DRMs) isolated from APP and PS-1 fibroblasts, compared to WT fibroblasts from healthy subjects. Modulation of raft GM1, including modest depletion of GM1 content and interference with GM1 exposure or negative charge, precluded the interaction of amyloid aggregates with the plasma membrane and the resulting cell damage in FAD fibroblasts and rat brains cortical neurons. These findings suggest a specific role for raft domains as primary mediators of amyloid toxicity in AD neurons.

Binding affinity of amyloid oligomers to cellular membranes is a generic indicator of cellular dysfunction in protein misfolding diseases

The conversion of peptides or proteins from their soluble native states into intractable amyloid deposits is associated with a wide range of human disorders. Misfolded protein oligomers formed during the process of aggregation have been identified as the primary pathogenic agents in many such conditions. Here, we show the existence of a quantitative relationship between the degree of binding to neuronal cells of different types of oligomers formed from a model protein, HypF-N, and the GM1 content of the plasma membranes. In addition, remarkably similar behavior is observed for oligomers of the Aβ42 peptide associated with Alzheimer’s disease. Further analysis has revealed the existence of a linear correlation between the level of the influx of Ca2+ across neuronal membranes that triggers cellular damage, and the fraction of oligomeric species bound to the membrane. Our findings indicate that the susceptibility of neuronal cells to different types of misfolded oligomeric assemblies is directly related to the extent of binding of such oligomers to the cellular membrane.