Cristina Cecchi

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.

Oxidative stress and reduced antioxidant defenses in peripheral cells from familial Alzheimer’s patients

We have measured the levels of typical end products of the processes of lipid peroxidation, protein oxidation, and total antioxidant capacity (TAC) in skin fibroblasts and lymphoblasts taken from patients with familial Alzheimers disease (FAD), sporadic Alzheimers disease (AD), and age-matched healthy controls. Compared to controls, the fibroblasts and lymphoblasts carrying amyloid precursor protein (APP) and presenilin-1 (PS-1) gene mutations showed a clear increase in lipoperoxidation products, malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE). In contrast, the antioxidant defenses of cells from FAD patients were lower than those from normal subjects. Lipoperoxidation and antioxidant capacity in lymphoblasts from patients affected by sporadic AD were virtually indistinguishable from the basal values of normal controls. An oxidative attack on protein gave rise to greater protein carbonyl content in FAD patients than in age-matched controls. Furthermore, ADP ribosylation levels of poly(ADP-ribose) polymerase (PARP) nuclear substrates were significantly raised, whereas the PARP content did not differ significantly between fibroblasts carrying gene mutations and control cells. These results indicate that peripheral cells carrying APP and PS-1 gene mutations show altered levels of oxidative markers even though they are not directly involved in the neurodegenerative process of AD. These results support the hypothesis that oxidative damage to lipid, protein, and DNA is an important early event in the pathogenesis of AD.

Gluthatione level is altered in lymphoblasts from patients with familial Alzheimer's disease

Intracellular levels of glutathione (GSH), glutathione disulphide (GSSG), glutamic acid and gamma-glutamyl cysteine synthetase (gamma-GCS) were measured in lymphoblast lines from patients with familial and sporadic Alzheimers disease (AD) and from age-matched controls. Lymphoblasts carrying presenilins (PS) and amyloid precursor protein (APP) genes mutations showed significantly decreased GSH content with respect to controls. Levels of GSSG and glutamic acid, as well as the activity of gamma-GCS were not significantly different in lymphoblasts carrying genes mutations as compared with control cells. These results indicate that even peripheral cells not involved in the neurodegenerative process of AD show altered GSH content when carrying PS and APP genes mutations. The provided data appear to be in accordance with the known alteration of GSH levels in central nervous system and strengthen the hypothesis of oxidative stress as an important, possibly crucial mechanism in the pathogenesis of AD.

Insights into the molecular basis of the differing susceptibility of varying cell types to the toxicity of amyloid aggregates

It has been reported that different tissue or cultured cell types are variously affected by the exposure to toxic protein aggregates, however a substantial lack of information exists about the biochemical basis of cell resistance or susceptibility to the aggregates. We investigated the extent of the cytotoxic effects elicited by supplementing the media of a panel of cultured cell lines with aggregates of HypF-N, a prokaryotic domain not associated with any amyloid disease. The cell types exposed to early, pre-fibrillar aggregates (not mature fibrils) displayed variable susceptibility to damage and to apoptotic death with a significant inverse relation to membrane content in cholesterol. Susceptibility to damage by the aggregates was also found to be significantly related to the ability of cells to counteract early modifications of the intracellular free Ca2+ and redox status. Accordingly, cell resistance appeared related to the efficiency of the biochemical equipment leading any cell line to sustain the activity of Ca2+ pumps while maintaining under control the oxidative stress associated with the increased metabolic rate. Our data depict membrane destabilization and the subsequent early derangement of ion balance and intracellular redox status as key events in targeting exposed cells to apoptotic death.

SIRT1 modulates MAPK pathways in ischemic-reperfused cardiomyocytes.

SIRT1, an ubiquitous NAD(+)-dependent deacetylase that plays a role in biological processes such as longevity and stress response, is significantly activated in response to reactive oxygen species (ROS) production. Resveratrol (Resv), an important activator of SIRT1, has been shown to exert major health benefits in diseases associated with oxidative stress. In ischemia–reperfusion (IR) injury, a major role has been attributed to the mitogen-activated protein kinase (MAPK) pathway, which is upregulated in response to a variety of stress stimuli, including oxidative stress. In neonatal rat ventricular cardiomyocytes subjected to simulated IR, the effect of Resv-induced SIRT1 activation and the relationships with the MAPK pathway were investigated. Resv-induced SIRT1 overexpression protected cardiomyocytes from oxidative injury, mitochondrial dysfunction, and cell death induced by IR. For the first time, we demonstrate that SIRT1 overexpression positively affects the MAPK pathway—via Akt/ASK1 signaling—by reducing p38 and JNK phosphorylation and increasing ERK phosphorylation. These results reveal a new protective mechanism elicited by Resv-induced SIRT1 activation in IR tissues and suggest novel potential therapeutic targets to manage IR-induced cardiac dysfunction.

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.

Seladin‐1/DHCR24 protects neuroblastoma cells against Aβ toxicity by increasing membrane cholesterol content

The role of brain cholesterol in Alzheimers disease (AD) is currently a matter of debate. Experimental evidence suggests that reducing circulating and brain cholesterol protects against AD, however recent data indicate that low membrane cholesterol results in neurode‐generation and that the cholesterol synthesis catalyst seladin‐1 is down‐regulated in AD‐affected brain regions. We previously reported a significant correlation between resistance to amyloid toxicity and content of membrane cholesterol in differing cultured cell types. Here we provide evidence that Aβ42 pre‐fibrillar aggregates accumulate more slowly and in reduced amount at the plasma membrane of human SH‐SY5Y neuroblastoma cells overexpressing seladin‐1 or treated with PEG‐cholesterol than at the membrane of control cells. The accumulation was significantly increased in cholesterol‐depleted cells following treatment with the specific seladin‐1 inhibitor 5,22E‐cholestadien‐3‐ol or with methyl‐β‐cyclodextrin. The resistance to amyloid toxicity and the early cytosolic Ca2+ rise following exposure to Aβ42 aggregates were increased and prevented, respectively, by increasing membrane cholesterol whereas the opposite effects were found in cholesterol‐depleted cells. These results suggest that seladin‐1‐dependent cholesterol synthesis reduces membrane‐aggregate interaction and cell damage associated to amyloid‐induced imbalance of cytosolic Ca2+. Our findings extend recently reported data indicating that seladin‐1 overexpression directly enhances the resistance to Aβ toxicity featuring seladin‐1/DHCR 24 as a possible new susceptibility gene for sporadic AD.

The amyloid-cell membrane system. The interplay between the biophysical features of oligomers/fibrils and cell membrane defines amyloid toxicity.

Amyloid cytotoxicity, structure and polymorphisms are themes of increasing importance. Present knowledge considers any peptide/protein able to undergo misfolding and aggregation generating intrinsically cytotoxic amyloids. It also describes growth and structure of amyloid fibrils and their possible disassembly, whereas reduced information is available on oligomer structure. Recent research has highlighted the importance of the environmental conditions as determinants of the amyloid polymorphisms and cytotoxicity. Another body of evidence describes chemical or biological surfaces as key sites of protein misfolding and aggregation or of interaction with amyloids and the resulting biochemical modifications inducing cell functional/viability impairment. In particular, the membrane lipid composition appears to modulate cell response to toxic amyloids, thus contributing to explain the variable vulnerability to the same amyloids of different cell types. Finally, a recent view describes amyloid toxicity as an emerging property dependent on a complex interplay between the biophysical features of early aggregates and the interacting cell membranes taken as a whole system.

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.