Mariagioia Zampagni

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.

Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein oligomers

Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligomers preformed from three different proteins added extracellularly to cultured cells. All the chaperones were found to decrease oligomer toxicity significantly, even at very low chaperone/protein molar ratios, provided that they were added extracellularly rather than being overexpressed in the cytosol. Infrared spectroscopy and site-directed labeling experiments using pyrene ruled out structural reorganizations within the discrete oligomers. Rather, confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and chaperones. Moreover, atomic force microscopy imaging indicated that larger assemblies of oligomers are formed in the presence of the chaperones. This suggests that the chaperones bind to the oligomers and promote their assembly into larger species, with consequent shielding of the reactive surfaces and a decrease in their diffusional mobility. Overall, the data indicate a generic ability of chaperones to neutralize extracellular misfolded oligomers efficiently and reveal that further assembly of protein oligomers into larger species can be an effective strategy to neutralize such extracellular species.

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.

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.

Large Proteins Have a Great Tendency to Aggregate but a Low Propensity to Form Amyloid Fibrils

The assembly of soluble proteins into ordered fibrillar aggregates with cross-β structure is an essential event of many human diseases. The polypeptides undergoing aggregation are generally small in size. To explore if the small size is a primary determinant for the formation of amyloids under pathological conditions we have created two databases of proteins, forming amyloid-related and non-amyloid deposits in human diseases, respectively. The size distributions of the two protein populations are well separated, with the systems forming non-amyloid deposits appearing significantly larger. We have then investigated the propensity of the 486-residue hexokinase-B from Saccharomyces cerevisiae (YHKB) to form amyloid-like fibrils in vitro. This size is intermediate between the size distributions of amyloid and non-amyloid forming proteins. Aggregation was induced under conditions known to be most effective for amyloid formation by normally globular proteins: (i) low pH with salts, (ii) pH 5.5 with trifluoroethanol. In both situations YHKB aggregated very rapidly into species with significant β-sheet structure, as detected using circular dichroism and X-ray diffraction, but a weak Thioflavin T and Congo red binding. Moreover, atomic force microscopy indicated a morphology distinct from typical amyloid fibrils. Both types of aggregates were cytotoxic to human neuroblastoma cells, as indicated by the MTT assay. This analysis indicates that large proteins have a high tendency to form toxic aggregates, but low propensity to form regular amyloid in vivo and that such a behavior is intrinsically determined by the size of the protein, as suggested by the in vitro analysis of our sample protein.

Protective effect of new S-acylglutathione derivatives against amyloid-induced oxidative stress

Recent data support the role of oxidative stress in the pathogenesis of Alzheimer disease (AD). In particular, glutathione (GSH) metabolism is altered and its levels are decreased in affected brain regions and peripheral cells from AD patients and in experimental models of AD. In the past decade, interest in the protective effects of various antioxidants aimed at increasing intracellular GSH content has been growing. Because much experimental evidence suggests a possible protective role of unsaturated fatty acids in age-related diseases, we designed the synthesis of new S-acylglutathione (acyl-SG) thioesters. S-Lauroylglutathione (lauroyl-SG) and S-palmitoleoylglutathione (palmitoleoyl-SG) were easily internalized into the cells and they significantly reduced Abeta42-induced oxidative stress in human neurotypic SH-SY5Y cells. In particular, acyl-SG thioesters can prevent the impairment of intracellular ROS scavengers, intracellular ROS accumulation, lipid peroxidation, and apoptotic pathway activation. Palmitoleoyl-SG seemed more effective in cellular protection against Abeta-induced oxidative damage than lauroyl-SG, suggesting a valuable role for the monounsaturated fatty acid. In this study, we demonstrate that acyl-SG derivatives completely avoid the sharp lipoperoxidation in primary fibroblasts from familial AD patients occurring after exposure to Abeta42 aggregates. Hence, we put forward these derivatives as new antioxidant compounds which could be excellent candidates for therapeutic treatment of AD and other oxidative stress-related 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.

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.