Gianfranco Liguri

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.

Cholesterol in Alzheimer's Disease: Unresolved Questions

The role of cholesterol as a susceptibility factor or a protective agent in neurodegeneration and, more generally, in amyloid-induced cytotoxicity is still controversial. Epidemiological studies on the hypercholesterolemia-AD risk relation and some reports indicating a beneficial effect of statin therapy suggest cholesterol as a susceptibility factor in AD. The ApoE4 genotype as a prevalent genetic risk factor for AD and the function of ApoE as main cholesterol carrier in the brain also underlie a close cholesterol load-AD risk relation. Finally, cell biology evidences support a critical involvement of lipid raft cholesterol in the modulation of beta- and gamma-secretase cleavage of APP with altered Abeta production. However, little exchange does exist between circulating and brain cholesterol, the latter arising from endogenous synthesis. In addition, increasing evidence supports the idea that amyloid cytotoxicity in most cases is initiated by oligomer recruitment at the cell membrane with loss of membrane integrity, Ca(2+) ingress into the cell, oxidative stress and apoptosis. In such a scenario, increased membrane cholesterol seems to be protective by disfavouring aggregate binding to the membrane. Recent findings also indicate that a reduction of cellular cholesterol favours co-localization of BACE1 and APP in non-raft membrane domains and hinders generation of plasmin, an Abeta-degrading enzyme. Finally, recent researches on Seladin-1, involved in cholesterol biosynthesis, show that modulation of membrane cholesterol affects Abeta generation and cell resistance against Abeta oligomer toxicity. These data confirm previous findings indicating a reduction of the cholesterol/phospholipid ratio in aged and AD brains. The aim of this review is to critically discuss some of the main results reported in the recent years in this field supporting a role of cholesterol either as a susceptibility factor or as a protective agent in 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.

Changes in Na+,K-ATPase, Ca2-ATPase and some soluble enzymes related to energy metabolism in brains of patients with Alzheimer's disease

Hexokinase, lactate dehydrogenase, acylphosphatase, (Na+,K+)-ATPase and Ca2(+)-ATPase of selected areas from postmortem Alzheimers disease brains were studied. Hexokinase and lactate dehydrogenase were significantly changed in all the examined subcortical nuclei. (Na+,K+)-ATPase activity was altered in several areas of Alzheimers disease brains. No changes in Ca2(+)-ATPase and acylphosphatase were observed. The main alterations of the assayed enzymes were observed in subcortical areas but not in cortical areas of Alzheimers disease brains.

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.

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.

Increased susceptibility to amyloid toxicity in familial Alzheimer's fibroblasts.

Much experimental evidence suggests that an imbalance in cellular redox status is a major factor in the pathogenesis of Alzheimers disease (AD). Our previous data showed a marked increase in membrane lipoperoxidation in primary fibroblasts from familial AD (FAD) patients. In the present study, we demonstrate that when oligomeric structures of Abeta 1-40 and Abeta 1-42 are added to the culture media, they accumulate quicker near the plasma membrane, and are internalized faster and mostly in APPV717I fibroblasts than in age-matched healthy cells; this results in an earlier and sharper increase in the production of reactive oxygen species (ROS). Higher ROS production leads in turn to an increase in membrane oxidative-injury and significant impairment of cellular antioxidant capacity, giving rise to apoptotic cascade activation and finally to a necrotic outcome. In contrast, healthy fibroblasts appear more resistant to amyloid oxidative-attack, possibly as a result of their plasma membrane integrity and powerful antioxidant capacity. Our data are consistent with increasing evidence that prefibrillar aggregates, compared to mature fibrils, are likely the more toxic species of the peptides. These findings provide compelling evidence that cells bearing increased membrane lipoperoxidation are more susceptible to aggregate toxicity as a result of their reduced ability to counteract amyloid oligomeric attack.

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.

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.