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The aggregation and deposition of misfolded β-amyloid peptide (Aβ42) is a consistent pathological hallmark of Alzheimer Disease (AD). Mounting pieces of evidence indicate that the auto-assembly of As42 into toxic oligomers and fibrillar aggregates is a multi-steps process that causes an increase of ROS (Reactive oxygen species) level, disruption of calcium homeostasis and altered membrane integrity leading to neuronal death. Oxidation is thought to promote As42 aggregation, so the use of natural dietary antioxidants that can interfere or inhibit As aggregation and its cytotoxicity is a promising approach. In this study, we investigate a panel of extracts from eight Tunisian medicinal plants for their ability to counteract amyloid aggregation. A screening assay was developed to identify and to rank extracts that hinder Aβ aggregation. Thus, we report that secondary metabolites extracted from Lawsonia inermis L., Punica granatum L. and Pistacia lentiscus L. hinder Aβ42 aggregation during the early secondary structure transitions to amyloid by inhibiting the conformational changes to a β sheet-rich structure, thus inhibiting its polymerization and fibrillogenisis. Cellular study allowed us to select the extract of L. inermis as the most bioactive extract; we showed that the extract of L. inermis reduces the toxicity, ROS and calcium levels induced by As42 aggregates. FRET analysis confirmed the ability of the extract of L. inermis to inhibit the interaction between Aβ42 aggregates and plasma membrane, thus reducing their cytotoxicity. These results suggested that methanolic extract from L. inermis could act as neuroprotective and therapeutic factors against toxic Aβ aggregates.

Amyloid-ß (Aß) fragments, oligomeric Aß aggregates, and pyroglutamylated-Aß peptides, as well as epigenetic mechanisms and autophagy dysfunction all appear to contribute in various ways to Alzheimer's disease progression. We previously showed that dietary supplementation of oleuropein aglycone, a natural phenol abundant in the extra virgin olive oil, can be protective by reducing Aß42 deposits in the brain of young and middle-aged TgCRND8 mice. Here, we extended our study to aged TgCRND8 mice showing increased pE3-Aß in the brain deposits. We report that oleuropein aglycone is active against glutaminylcyclase-catalyzed pE3-Aß generation reducing enzyme expression and interferes both with Aß42 and pE3-Aß aggregation. Moreover, the phenol astonishingly activates neuronal autophagy even in mice at advanced stage of pathology, where it increases histone 3 and 4 acetylation, which matches both a decrease of histone deacetylase 2 expression and a significant improvement of synaptic function. The occurrence of these functional, epigenetic, and histopathologic beneficial effects even at a late stage of the pathology suggests that the phenol could be beneficial at the therapeutic, in addition to the prevention, level.

A common molecular feature of amyloid neurodegenerative diseases is the unfolding/misfolding of specific proteins/peptides which consequently become prone to aggregate into toxic assemblies and deposits that are the key histopathological trait of these pathologies. Apart from the rare early-onset familiar forms, these neurodegenerative diseases are age-associated disorders whose symptoms appear in aged people after long incubation periods. This makes the therapeutic approach particularly compelling and boosts the search for both early diagnostic tools and preventive approaches. In this last respect, natural compounds commonly present in foods and beverages are considered promising molecules, at least on the bench side. The so-called 'nutraceutical approach' suggests life-long healthy diets, particularly focusing on food molecules that are candidates to enter clinical trials as such or following a targeted molecular engineering. Natural phenols abundant in 'healthy' foods such as extra virgin olive oil, red wine, green tea, red berries and spices, appear particularly promising.

Mediterranean and Asian diets are currently considered as the most healthy traditional feeding habits effective against risk of age-associated, particularly cardiovascular and neurodegenerative, diseases. A common feature of these two regimens is the abundance of foods and beverages of plant origin (green tea, extra virgin olive oil, red wine, spices, berries, and aromatic herbs) that are considered responsible for the observed beneficial effects. Epidemiological data suggest that the phenolic component remarkably enriched in these foods plays an important role in reducing the incidence of amyloid diseases, pathological conditions associated to tissue deposition of toxic protein aggregates responsible for progressive functional deterioration. Great effort is being spent to provide knowledge on the effects of several natural phenols in this context, moving from the test tube to animal models and, more slowly, to the patient's bed. An emerging feature that makes these molecules increasingly attractive for amyloid disease prevention and therapy is their wide spectrum of activity: recent pieces of evidence suggest that they can inhibit the production of amyloidogenic peptides from precursors, increase antioxidant enzyme activity, activate autophagy and reduce inflammation. Our concept should than shift from considering natural phenols simply as antioxidants or, at the best, as amyloid aggregation inhibitors, to describing them as potentially multitargeting drugs. A main concern is the low bioavailability of such compounds and efforts aimed at improving it are underway, with encapsulation strategies being the most promising ones.

Previous data have shown that oleuropein aglycone (OLE), the main secoiridoid phenol present in extra virgin olive oil, counteracts in vitro aggregation of the Aβ42 peptide and protects cultured cells and model organisms against aggregates toxicity. In this study we investigated the relative tissue toxicity of Aβ42 aggregated in vitro in the presence or in the absence of OLE by injecting the nucleus basalis magnocellularis (NBM) of adult male Wistar rats with a 1.5 μl solution containing OLE (450 μM) or Aβ42 (50 μM) aggregated in the absence (oligomers) or in the presence of 450 μM OLE. Control rats were injected with vehicle (1.5 μl). Thirty days after injection, the number of choline acetyltransferase (ChAT)-positive neurons, glia reaction and the Aβ peptide levels were detected by immunohistochemistry. An apparent reduction in the amount of soluble A11-positive oligomers was detected in the NBM injected with Aβ42 aggregated with OLE, as compared with the NBM injected with Aβ42 alone. In the latter case, the number of ChAT-positive neurons was significantly reduced (≈-33%) respect to that recorded in the NBM injected with phosphate buffer, OLE or Aβ42 aggregated with OLE. A markedly attenuated Aβ-induced astrocytes and microglia reaction was also found in the NBM injected with Aβ42 aggregated with OLE. Altogether, these data provide additional support to the anti-aggregation, neuroprotective and anti-inflammatory activities of this natural phenol, confirming its beneficial properties against neurodegeneration.

The presence of amyloid aggregates of human islet amyloid polypeptide (hIAPP), a hallmark of type 2 diabetes, contributes to pancreatic β-cell impairment, where oxidative stress plays a key role. A contribution of NADPH oxidase to reactive oxygen species (ROS) generation after cell exposure to micromolar concentrations of hIAPP aggregates has been suggested. However, little is known about β-cells exposure to lower amounts of hIAPP aggregates, similar to those found in human pancreas. Thus, we aimed to investigate the events resulting from RIN-5F cells exposure to nanomolar concentrations of toxic hIAPP aggregates. We found an early and transient rise of NADPH oxidase activity resulting from increased Nox1 expression following the engagement of receptor for advanced glycation end-products (RAGE) by hIAPP aggregates. Unexpectedly, NADPH oxidase activation was not accompanied by a significant ROS increase and the lipoperoxidation level was significantly reduced. Indeed, cell exposure to hIAPP aggregates affected the antioxidant defences, inducing a significant increase of the expression and activity of catalase and glutathione peroxidase. We conclude that exposure of pancreatic β-cells to nanomolar concentrations of hIAPP aggregates for a short time induces an hormetic response via the RAGE-Nox1 axis; the latter stimulates the enzymatic antioxidant defences that preserve the cells against oxidative stress damage., Graphical abstract, Highlights • Short time exposure of pancreatic β-cells to low hIAPP aggregate amounts is studied. • NADPH oxidase activity is increased after 3 h treatment with 60 nM hIAPP aggregates. • RAGE engagement by aggregates increases Nox1 expression. • Reduced lipoperoxidation and increased antioxidant enzymes were observed. • A protective hormetic response via RAGE-Nox1 is proposed.

Background: Translational research needs valid animal models of disease to discover new pathogenetic aspects and treatments. In Alzheimer's disease (AD), transgenic models are of great value for AD research and drug testing. Objective: It was the aim of this study to analyze the power of dietary polyphenols against neurodegeneration by investigating the effects of oleuropein aglycone (OLE), the main phenol in the extra virgin olive oil (EVOO), a key component of the Mediterranean diet (MD), in a mouse model of amyloid-β deposition. Methods: TgCRND8 mice (3.5 months old), expressing the mutant KM670/671NL+V717F h-βAPP695 transgene, and wild-type (wt) mice were used to study in vivo the effects of an 8-week dietary supplementation with OLE (50 mg/kg of diet) [Grossi et al: PLoS One 2013;8:e71702], following the European Communities Council Directive 86/609 (DL 116/92) and National Guidelines (permit number: 283/2012-B). Results: OLE administration ameliorates memory dysfunction, raises a significant autophagic response in the cortex and promotes the proliferation of newborn cells in the subgranular zone of the dentate gyrus of the hippocampus. Conclusions: Our findings support the beneficial effects of EVOO and highlight the possibility that continuous intake of high doses of OLE, both as a nutraceutical or as a food integrator, may prevent/delay the appearance of AD and reduce the severity of its symptoms.

Amyloid aggregation is a hallmark of several degenerative diseases affecting the brain or peripheral tissues, whose intermediates (oligomers, protofibrils) and final mature fibrils display different toxicity. Consequently, compounds counteracting amyloid aggregation have been investigated for their ability (i) to stabilize toxic amyloid precursors; (ii) to prevent the growth of toxic oligomers or speed that of fibrils; (iii) to inhibit fibril growth and deposition; (iv) to disassemble preformed fibrils; and (v) to favor amyloid clearance. Natural phenols, a wide panel of plant molecules, are one of the most actively investigated categories of potential amyloid inhibitors. They are considered responsible for the beneficial effects of several traditional diets being present in green tea, extra virgin olive oil, red wine, spices, berries and aromatic herbs. Accordingly, it has been proposed that some natural phenols could be exploited to prevent and to treat amyloid diseases, and recent studies have provided significant information on their ability to inhibit peptide/protein aggregation in various ways and to stimulate cell defenses, leading to identify shared or specific mechanisms. In the first part of this review, we will overview the significance and mechanisms of amyloid aggregation and aggregate toxicity; then, we will summarize the recent achievements on protection against amyloid diseases by many natural phenols.

Transthyretin (TTR) amyloidoses are familial or sporadic degenerative conditions that often feature heavy cardiac involvement. Presently, no effective pharmacological therapy for TTR amyloidoses is available, mostly due to a substantial lack of knowledge about both the molecular mechanisms of TTR aggregation in tissue and the ensuing functional and viability modifications that occur in aggregate-exposed cells. TTR amyloidoses are of particular interest regarding the relation between functional and viability impairment in aggregate-exposed excitable cells such as peripheral neurons and cardiomyocytes. In particular, the latter cells provide an opportunity to investigate in parallel the electrophysiological and biochemical modifications that take place when the cells are exposed for various lengths of time to variously aggregated wild-type TTR, a condition that characterizes senile systemic amyloidosis. In this study, we investigated biochemical and electrophysiological modifications in cardiomyocytes exposed to amyloid oligomers or fibrils of wild-type TTR or to its T4-stabilized form, which resists tetramer disassembly, misfolding, and aggregation. Amyloid TTR cytotoxicity results in mitochondrial potential modification, oxidative stress, deregulation of cytoplasmic Ca2+ levels, and Ca2+ cycling. The altered intracellular Ca2+ cycling causes a prolongation of the action potential, as determined by whole-cell recordings of action potentials on isolated mouse ventricular myocytes, which may contribute to the development of cellular arrhythmias and conduction alterations often seen in patients with TTR amyloidosis. Our data add information about the biochemical, functional, and viability alterations that occur in cardiomyocytes exposed to aggregated TTR, and provide clues as to the molecular and physiological basis of heart dysfunction in sporadic senile systemic amyloidosis and familial amyloid cardiomyopathy forms of TTR amyloidoses.

Growing evidence indicates that reactive oxygen species (ROS) may play a key role in human heart failure (HF). Monoamine oxidase (MAO) is emerging as a major ROS source in several cardiomyopathies. However, little is known about MAO activity in human failing heart and its relationship with redox imbalance. Therefore, we measured MAO activity in the left (LV) and in the right (RV) ventricle of human nonfailing (NF) and in end-stage ischemic (IHD) and nonischemic failing hearts. We found that both MAO isoforms (MAO-A/B) significantly increased in terms of activity and expression levels only in IHD ventricles. Catalase and aldehyde dehydrogenase-2 activities (ALDH-2), both implicated in MAO-catalyzed catecholamine catabolism, were significantly elevated in the failing LV, whereas, in the RV, statistical significance was observed only for ALDH-2. Oxidative stress markers levels were significantly increased only in the failing RV. Actin oxidation was significantly elevated in both failing ventricles and related to MAO-A activity and to functional parameters. These data suggest a close association between MAO-A-dependent ROS generation, actin oxidation, and ventricular dysfunction. This latter finding points to a possible pathogenic role of MAO-A in human myocardial failure supporting the idea that MAO-A could be a new therapeutic target in HF.

Amyloid aggregation starts with the initial misfolding of peptide/protein precursors, with subsequent structural rearrangement into oligomers and protofibrils; the latter eventually organize into fibrils with shared basic structural features, found deposited in amyloid diseases. Mounting evidence indicates early oligomers as the most toxic amyloid species; accordingly, the search of inhibitors of their growth is considered a promising target to prevent amyloid toxicity. We recently showed that oleuropein aglycon, a polyphenol abundant in the extra virgin olive oil, interferes with the aggregation of amylin (involved in type-2 diabetes), eliminating its cytotoxicity. Here we report that oleuropein aglycon also hinders amyloid aggregation of Aβ(1-42) and its cytotoxicity, suggesting a general effect of such polyphenol. In particular, by using a wide panel of different spectroscopic, immunologic, cell viability and imaging techniques we provide a more detailed description of Aβ(1-42) structural modifications arising in the presence of the inhibitor and the resulting cytotoxicity. We here report that the polyphenol eliminates the appearance of early toxic oligomers favouring the formation of stable harmless protofibrils, structurally different from the typical Aβ(1-42) fibrils. We also show that oleuropein aglycon is maximally effective when is present at the beginning of the aggregation process; furthermore, when added to preformed fibrils, it does not induce the release of toxic oligomers but, rather, neutralizes any residual toxicity possibly arising from the residual presence of traces of soluble oligomers and other toxic aggregates. The possible use of this polyphenol as anti-aggregation molecule is discussed in the light of these data.

Pancreatic amyloid deposits of amylin are a hallmark of Type II diabetes and considerable evidence indicates that amylin oligomers are cytotoxic to beta-cells. Many efforts are presently spent to find out naturally occurring molecules, or to design synthetic ones, able to hinder amylin aggregation or to protect cells against aggregate cytotoxicity. In this context, a protective effect of some polyphenols against amyloid cytotoxicity was reported. Actually dietary polyphenols are endowed with multiple health benefits, and extra virgin olive oil is attracting increasing interest as a source of these substances. Here, we investigated the effects on amylin aggregation and cytotoxicity of the secoiridoid oleuropein aglycon, the main phenolic component of extra virgin olive oil. We found that oleuropein, when present during the aggregation of amylin, consistently prevented its cytotoxicity to RIN-5F pancreatic beta-cells, as determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide test and caspase-3 activity assay. A lack of interaction with the cell membrane of amylin aggregates grown in the presence of oleuropein was shown by fluorescence microscopy and synthetic lipid vesicle permeabilization. Moreover, our ThT assay, circular dichroism analysis and electron microscopy images suggested that oleuropein interferes with amylin aggregation, resulting in a different path skipping the formation of toxic pre-fibrillar aggregates. These results provide a molecular basis for some of the benefits potentially coming from extra virgin olive oil consumption and pave the way to further studies on the possible pharmacological use of oleuropein to prevent or to slow down the progression of type II diabetes.

Activation of human platelets by cross-linking of the low-affinity receptor for immunoglobulin G (FcγRIIA) is initiated by Src kinase–mediated phosphorylation of the immunoreceptor tyrosine–based activation motif (ITAM) within the receptor, but the identity of the enzyme responsible for its dephosphorylation and inactivation is unknown. Here we report that the 18-kDa low-molecular-weight phosphotyrosine phosphatase (LMW-PTP) is expressed in human platelets and undergoes subcellular redistribution upon FcγRIIA cross-linking. In vitro, LMW-PTP was found to efficiently dephosphorylate activated FcγRIIA and LAT, but not Syk or phospholipase Cγ2. In the megakaryocytic cell line DAMI, antibody-induced phosphorylation of FcγRIIA was rapid and transient. The late dephosphorylation of FcγRIIA was dramatically delayed upon reduction of LMW-PTP expression by siRNA. Strikingly, overexpression of LMW-PTP resulted in the inhibition of antibody-induced phosphorylation of FcγRIIA, and caused a more rapid dephosphorylation. In addition, overexpression of LMW-PTP inhibited activation of Syk downstream of FcγRIIA and reduced intracellular Ca2+ mobilization. These results demonstrate that LMW-PTP is responsible for FcγRIIA dephosphorylation, and is implicated in the down-regulation of cell activation mediated by this ITAM-bearing immunoreceptor.

Several human degenerative diseases involve amyloidogenic peptides/proteins with high conformational plasticity and propensity to self-aggregate into polymeric fibrillar assemblies sharing the cross-β structure and endowed with cytotoxic potential. Although the mechanisms of amyloid growth and toxicity are not fully understood, a common property of amyloids is their ability to interact with lipid bilayers disturbing membrane integrity. Lipid bilayers can also act as conformational catalysts, favoring protein misfolding and inducing the growth of aggregation nuclei, early oligomers, and mature fibrils with specific biophysical, structural, and toxicity features. This Perspective will highlight these effects in the context of a membrane-oligomer system where the conformational/biophysical features of either component affect those of the other. In this context, we will highlight the modulation of the protein-cell surface interaction by the content of membrane cholesterol and gangliosides, notably GM1. In particular, we will discuss data that indicate how these interactions affect the structural and stability properties of both protein and bilayers as well as the final cytotoxic effect. Our goal is to propose shared membrane-based mechanisms that could apply to any amyloidogenic peptide/protein, providing a biochemical background for amyloid growth and toxicity.

The healthy effects of plant polyphenols, some of which characterize the so-called Mediterranean diet, have been shown to arise from epigenetic and biological modifications resulting, among others, in autophagy stimulation. Our previous work highlighted the beneficial effects of oleuropein aglycone (OLE), the main polyphenol found in the extra virgin olive oil, against neurodegeneration both in cultured cells and in model organisms, focusing, in particular, autophagy activation. In this study we investigated more in depth the molecular and cellular mechanisms of autophagy induction by OLE using cultured neuroblastoma cells and an OLE-fed mouse model of amylod beta (Aβ) deposition. We found that OLE triggers autophagy in cultured cells through the Ca2+-CAMKKβ-AMPK axis. In particular, in these cells OLE induces a rapid release of Ca2+ from the SR stores which, in turn, activates CAMKKβ, with subsequent phosphorylation and activation of AMPK. The link between AMPK activation and mTOR inhibition was shown in the OLE-fed animal model in which we found that decreased phospho-mTOR immunoreactivity and phosphorylated mTOR substrate p70 S6K levels match enhanced phospho-AMPK levels, supporting the idea that autophagy activation by OLE proceeds through mTOR inhibition. Our results agree with those reported for other plant polyphenols, suggesting a shared molecular mechanism underlying the healthy effects of these substances against ageing, neurodegeneration, cancer, diabetes and other diseases implying autophagy dysfunction.

Amyloid diseases are characterized by the deposition of typically aggregated proteins/peptides in tissues, associated with degeneration and progressive functional impairment. Alzheimer's disease is one of the most studied neurodegenerative amyloid diseases and, in Western countries, a significant cause of dementia in the elderly. The so-called "Mediterranean diet" has been considered for long as the healthier dietary regimen, characterised by a great abundance in vegetables and fruits, extra virgin olive oil as the main source of fat, a moderate consumption of red wine and a reduced intake of proteins from red meat. Recent epidemiological studies support the efficacy of the Mediterranean diet not only against cardiovascular and cancer diseases (as previously demonstrated) but also against the cognitive decline associated with ageing, and several data are highlighting the role played by natural phenols, of which red wine and extra virgin olive oil are rich, in such context. In the meantime, studies conducted both in vivo and in vitro have started to reveal the great potential of the phenolic component of extra virgin olive oil (mainly oleuropein aglycone and oleocanthal) in counteracting amyloid aggregation and toxicity, with a particular emphasis on the pathways involved in the onset and progression of Alzheimer's disease: amyloid precursor protein processing, amyloid-beta (Aβ) peptide and tau aggregation, autophagy impairment, neuroinflammation. The aim of this review is to summarize the results of such research efforts, showing how the action of these phenols goes far beyond their renowned antioxidant activity and revealing their potential as multi-targeting agents against Alzheimer's disease.

The amyloid plaques and neurofibrillary tangles found in the Alzheimer's disease (AD) brain arise as a result of self-assembly into fibrillar material of amyloid-β protein (Aβ) and hyperphosphorylated tau, respectively, through a pathological process starting with the appearance of aggregation nuclei and neurotoxic oligomers. Accordingly, the search of inhibitors of oligomer nucleation and growth is considered a promising target to prevent amyloid toxicity. In recent years, a number of dietary factors including antioxidants, vitamins, and polyphenols have been characterized for their ability to protect cells stressed by several factors including the presence of amyloid deposits as well as to inhibit amyloid self-assembly and cytotoxicity and some of them are currently in clinical trial. The present review summarizes the findings on the beneficial effects against neurodegeneration and other peripheral inflammatory and degenerative diseases of oleuropein aglycone (OLE), a natural phenol abundant in the extra virgin olive oil. The data presently available suggest that OLE could provide a protective and therapeutic effect against a number of pathologies, including AD as well as obesity, type 2 diabetes, non-alcoholic hepatitis, and other natural or experimentally-induced pathological conditions. Such a protection could result, at least in part, in a remarkable improvement of the pathological signs arising from stress conditions including oxidative stress, an excessive inflammatory response, and the presence of cytotoxic aggregated material. In particular, the recent data on the cellular and molecular correlates of OLE neuroprotection suggest it could also play a therapeutic role against AD.

Amyloid diseases are characterized by the deposition of typically aggregated proteins/peptides in tissues, associated with degeneration and progressive functional impairment. Alzheimer’s disease is one of the most studied neurodegenerative amyloid diseases and, in Western countries, a significant cause of dementia in the elderly. The so-called “Mediterranean diet” has been considered for long as the healthier dietary regimen, characterised by a great abundance in vegetables and fruits, extra virgin olive oil as the main source of fat, a moderate consumption of red wine and a reduced intake of proteins from red meat. Recent epidemiological studies support the efficacy of the Mediterranean diet not only against cardiovascular and cancer diseases (as previously demonstrated) but also against the cognitive decline associated with ageing, and several data are highlighting the role played by natural phenols, of which red wine and extra virgin olive oil are rich, in such context. In the meantime, studies conducted both in vivo and in vitro have started to reveal the great potential of the phenolic component of extra virgin olive oil (mainly oleuropein aglycone and oleocanthal) in counteracting amyloid aggregation and toxicity, with a particular emphasis on the pathways involved in the onset and progression of Alzheimer’s disease: amyloid precursor protein processing, amyloid-beta (Aβ) peptide and tau aggregation, autophagy impairment, neuroinflammation. The aim of this review is to summarize the results of such research efforts, showing how the action of these phenols goes far beyond their renowned antioxidant activity and revealing their potential as multi-targeting agents against Alzheimer’s disease.

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.

Hematopoietic cells, particularly megakaryoblastic ones, display a high level of low M r phosphotyrosine protein phosphatase (LMW-PTP) expression; nevertheless, the role of this PTP in such cellular lineages has been scarcely investigated. Here, we demonstrate that LMW-PTP is able to associate and dephosphorylate signal transducer and activator of transcription-5 (STAT5) in DAMI megakaryocytic cells. Numerous researchers repeatedly hypothesized the association of a regulatory phosphotyrosine protein phosphatase with STAT5 C-terminus, but such phosphotyrosine protein phosphatase remained unknown. We show evidence indicating that the association of STAT5 and LMW-PTP does not exclusively involve the phosphatase active site and phosphotyrosine residue of STAT5, and we individuate an essential region of interaction at STAT5 C-terminus, coinciding with the previously hypothesized PTP-associating domain.

Low M r phosphotyrosine protein phosphatase is a cytosolic enzyme which dephosphorylates platelet-derived growth factor and insulin receptor in vivo, thus reducing cellular mitogenic response to such growth factors. Following cell stimulation with platelet-derived growth factor the phosphatase undergoes a redistribution from the citosol to the Triton X-100-insoluble fraction where its activity upon the growth factor receptor is intense. Previous research uncovered evidence that low M r phosphotyrosine protein phosphatase dephosphorylates the epidermal growth factor receptor in vitro. Here we demonstrate that in vivo the enzyme is not active on the phosphorylated epidermal growth factor receptor and it does not influence the mitogenic response of cells. Since the enzyme distribution is not affected by epidermal growth factor stimulation, involvement of a recruitment mechanism in the definition of low M r phosphotyrosine protein phosphatase substrate specificity is hypothesized.

Low Mr phosphotyrosine protein phosphatase (PTP) is a cytosolic enzyme whose activity upon platelet-derived growth factor (PDGF) and insulin receptors has been demonstrated in vivo. In our study we demonstrate that this enzyme, both naturally expressed and overexpressed in NIH/3T3 fibroblasts, translocates from the cytosol to the Triton X-100 insoluble fraction following stimulation with PDGF. It emerges that the phosphorylation of a defined population of PDGF receptors, which is localized in this fraction and seems to be endowed with peculiar features and functions, is particularly affected by low Mr PTP overexpression.

Recent studies show that glutathione, while being involved in the well-known physiological processes of amino acid transport and detoxification, can also play a part in cell proliferation events. Cell treatment with l-buthionine sulphoximine, which causes glutathione depletion, is accompanied by a decrease in cell proliferation. At present no precise relationship between this thiol and any critical intermediate of the mitogenic cascade has been proved. In this study, conducted on NIH/3T3 murine fibroblasts, we demonstrate a strict correlation between glutathione levels and platelet-derived growth-factor-receptor activation in response to stimulation and cell proliferation. The receptor autophosphorylation is severely impaired at low glutathione cellular levels. The interaction of glutathione with this growth-factor receptor in vivo, while being rather specific, is complex and may involve both cytosolic and extracellular receptor domains.

The level of both isoforms of acylphosphatase was evaluated in the human erythroleukemia K562 cell line during differentiation. K562 cells were treated with PMA, which induces megakaryocytic differentiation, and with aphidicolin or hemin, which stimulate erythrocytic differentiation. While the MT isoform showed an average 10-fold increase independently of the differentiating agent used, only hemin treatment caused a similar increase of the CT isoform, suggesting a different role of the two isoforms in the cell. Treatment with either hemin or aphidicolin of K562 cells overexpressing the two acylphosphatase isoforms suggested the possibility that acylphosphatases play a role in the onset of differentiation.

The role of low M r phosphotyrosine protein phosphatase (PTPase) in the control of cell proliferation was studied. A synthetic gene coding for PTPase was transfected and expressed in NIH/3T3 fibroblasts. The effects of the enzyme were particularly evident when cells were stimulated by platelet-derived growth factor (PDGF). The mitogenic response to PDGF was decreased and the inhibition reached 90%. This effect was more pronounced with respect to fetal calf serum stimulation. Hormone-dependent autophosphorylation of the PDGF receptor was significantly reduced. These results demonstrate that low M r PTPase, a cytosolic enzyme, not only affects cellular response to PDGF but also reduces the membrane receptor autophosphorylation.

Amyloid aggregation starts with the initial misfolding of peptide/protein precursors, with subsequent structural rearrangement into oligomers and protofibrils; the latter eventually organize into fibrils with shared basic structural features, found deposited in amyloid diseases. Mounting evidence indicates early oligomers as the most toxic amyloid species; accordingly, the search of inhibitors of their growth is considered a promising target to prevent amyloid toxicity. We recently showed that oleuropein aglycon, a polyphenol abundant in the extra virgin olive oil, interferes with the aggregation of amylin (involved in type-2 diabetes), eliminating its cytotoxicity. Here we report that oleuropein aglycon also hinders amyloid aggregation of Aβ(1-42) and its cytotoxicity, suggesting a general effect of such polyphenol. In particular, by using a wide panel of different spectroscopic, immunologic, cell viability and imaging techniques we provide a more detailed description of Aβ(1-42) structural modifications arising in the presence of the inhibitor and the resulting cytotoxicity. We here report that the polyphenol eliminates the appearance of early toxic oligomers favouring the formation of stable harmless protofibrils, structurally different from the typical Aβ(1-42) fibrils. We also show that oleuropein aglycon is maximally effective when is present at the beginning of the aggregation process; furthermore, when added to preformed fibrils, it does not induce the release of toxic oligomers but, rather, neutralizes any residual toxicity possibly arising from the residual presence of traces of soluble oligomers and other toxic aggregates. The possible use of this polyphenol as anti-aggregation molecule is discussed in the light of these data.

STATs are involved in a variety of cellular processes, including cell proliferation and differentiation. They are activated through tyrosine phosphorylation, which promotes their dimerization via SH2 domains. We have demonstrated previously that in DAMI megakaryoblastic cells LMW-PTP dephosphorylates STAT5, interacting with an essential sequence of nine amino acids in its C-terminal region. Here we characterize STAT5 tyrosine phosphorylation and its interaction with LMW-PTP during early phorbol-12-myristate-13-acetate-induced megakaryocyte differentiation; these processes show clear dependence on STAT5 threonine phosphorylation. Since protein kinase C inhibition prevents phorbol-12-myristate-13-acetate-induced STAT5 threonine phosphorylation and association with LMW-PTP, it follows that these processes depend on protein kinase C activity. By using a Thr757/Val mutant of STAT5 we also demonstrate that the 757 serine/threonine conserved residue, which is in the STAT5A region involved in the interaction with LMW-PTP, is essential for such an association, though its phosphorylation is not necessary.

The finding that aggregates of disease-unrelated proteins can induce cell death indicates that proteins may act as potential toxins. Cells experiencing toxic protein aggregates often display modifications of the redox status and ion balance, eventually leading to apoptosis; however, in some cases, tissue and cultured cell types die with features of necrosis. To elucidate the pathways leading to such different outcomes, we studied the biochemical features of death in H-END and NIH/3T3 cells exposed to prefibrillar aggregates of a disease-unrelated protein. The two types of cells died by apoptosis and necrosis, respectively. The pattern of caspase and proapoptotic factor activation was investigated together with the extent of mitochondria impairment and the energy load in either cell line. Our data depict a scenario where the events related to the extrinsic pathway of apoptosis are the same in the two cell lines, the difference in the final outcome being related to the extent of mitochondria derangement, possibly due to the different ability of the cells to counteract ion homeostasis impairment.

Low M(r) phosphotyrosine protein phosphatase interferes in vivo with the activation of several growth factor receptors and is transiently redistributed, following cell stimulation with platelet-derived growth factor, from the cytosol to the cytoskeleton. We demonstrate here that this phosphatase also participates in the regulation of cell spreading and migration, pointing to its involvement in cytoskeleton organization. Low M(r) phosphotyrosine protein phosphatase-overexpressing fibroblasts are, indeed, less spread than controls and display a significantly decreased number of focal adhesions and increased cell motility. Furthermore, p125 focal adhesion kinase is associated to, and dephosphorylated by, low M(r) phosphotyrosine protein phosphatase both in vitro and in vivo. This event is consistent with an altered association of pp60(src) with focal adhesion kinase. The activation of extracellular signal-regulated kinase, another well known event downstream of the focal adhesion kinase, is also affected. On the other hand, cells overexpressing the dominant-negative form of low M(r) phosphotyrosine protein phosphatase exhibit hyperphosphorylated focal adhesion kinase, reduced motility, and an increased number of focal adhesions, which are distributed all over the ventral cell surface. Taken together, the results reported here are in keeping with low M(r) phosphotyrosine protein phosphatase participation in FAK-mediated focal adhesion remodeling.

Fibroblast growth factor receptor (class IV) shares a certain degree of similarity with class III members like platelet- derived growth factor and macrophage-colony-stimulating factor receptors, which, once activated, are substrates of low Mr phosphotyrosine protein phosphatase. Up until now no phospho- tyrosine phosphatase has been shown to act on this receptor in vivo. Here we demonstrate that low Mr phosphotyrosine protein phosphatase is able to reduce receptor tyrosine phosphorylation and cell proliferation in response to basic fibroblast growth factor. Contrary to what was previously observed for platelet- derived growth factor, during cell stimulation with basic fibroblast growth factor, no enzyme redistribution among cellular compartments is observed. z 1999 Federation of European Biochemical Societies. growth factor receptor (EGFr) (class I receptor) has been previously evidenced in vitro (9), but we showed that this has no correspondence in vivo (10). We recently demonstrated that low Mr PTP undergoes a redistribution from the cytosol to the Triton X-100-insoluble fraction following cell stimulation with PDGF, and that in this fraction low Mr PTP exerts most of its activity on PDGFr (4). This phenomenon, in contrast, was not observed with EGF and, correspondingly, no eiect on EGFr activation was observed (10). These ¢ndings, taken together with the fact that EGFr is a substrate for this PTP in vitro, i.e. in a condition where the necessity of enzyme compartmentaliza- tion is overcome, seemed to stress the importance of intra- cellular localization of low Mr PTP in the de¢nition of en- zyme speci¢city in vivo. The high a⁄nity ¢broblast growth factor receptor (FGFr) family includes several members which bind more than one type of FGF. These receptors, though being allocated to a separate class (class IV), share several structure similarities with the members of class III, such that the ¢rst identi¢ed gene encoding FGFr was called £g, which stands for 'fms- like gene', where fms is the gene encoding M-CSFr. Here we demonstrate that these similarities extend also to the susceptibility of FGFr to low Mr PTP, as both receptor phosphorylation and mitogenic response elicited by the bind- ing of basic FGF (bFGF) were aiected by phosphatase over- expression. To our knowledge, this represents the ¢rst re- ported eiect of a tyrosine phosphatase on FGFr phosphorylation. When we veri¢ed the connection between the activity of low Mr PTP and its translocation in response to M-CSF or bFGF, no redistribution of the phosphatase was observed.

The claimed beneficial effects of the Mediterranean diet include prevention of several age-related dysfunctions including neurodegenerative diseases and Alzheimer-like pathology. These effects have been related to the protection against cognitive decline associated with aging and disease by a number of polyphenols found in red wine and extra virgin olive oil. The double transgenic TgCRND8 mice (overexpressing the Swedish and Indiana mutations in the human amyloid precursor protein), aged 1.5 and 4, and age-matched wild type control mice were used to examine in vivo the effects of 8 weeks dietary supplementation of oleuropein aglycone (50 mg/kg of diet), the main polyphenol found in extra virgin olive oil. We report here that dietary supplementation of oleuropein aglycone strongly improves the cognitive performance of young/middle-aged TgCRND8 mice, a model of amyloid-ß deposition, respect to age-matched littermates with un-supplemented diet. Immunofluorescence analysis of cerebral tissue in oleuropein aglycone-fed transgenic mice showed remarkably reduced ß-amyloid levels and plaque deposits, which appeared less compact and "fluffy"; moreover, microglia migration to the plaques for phagocytosis and a remarkable reduction of the astrocyte reaction were evident. Finally, oleuropein aglycone-fed mice brain displayed an astonishingly intense autophagic reaction, as shown by the increase of autophagic markers expression and of lysosomal activity. Data obtained with cultured cells confirmed the latter evidence, suggesting mTOR regulation by oleuropein aglycone. Our results support, and provide mechanistic insights into, the beneficial effects against Alzheimer-associated neurodegeneration of a polyphenol enriched in the extra virgin olive oil, a major component of the Mediterranean diet.

Having determined the complete amino acid sequence of a cytosolic phosphatase purified from bovine liver, we studied the role of this enzyme (referred to as ‘PTPase’) in the control of cell proliferation. We used NIH/3T3 fibroblasts, both normal and transformed by the oncogenes v-erbB, v-src, and v-raf: a synthetic gene coding for PTPase was transfected into, and overexpressed in, normal and transformed NIH/3T3 cells with resulting inhibition of cell growth. Inhibition of proliferation correlated with the level of foreign PTPase; growth in soft agar was also inhibited in transformants overexpressing the enzyme. However, PTPase overexpression did not inhibit the rapid turnover of inositol lipids stimulated by platelet-derived growth factor. We conclude that this novel PTPase is active on cell type-specific signalling substrates that control normal and transformed fibroblast proliferation.

We measured the level of reduced glutathione (GSH) and oxidized glutathione (GSSG) in normal and oncogene-transformed NIH/3T3 fibroblasts and 32D hematopoietic cells. NIH/3T3 cells transformed by the activated oncogenes erbB, src, and raf, showed increased levels of GSH with concomitant alterations in the levels of GSH-related enzymes. Transfection and over-expression of a synthetic gene coding for a phosphotyrosine protein phosphatase (PTPase), which inhibited the proliferation of normal and transformed NIH/3T3 cells, was accompanied by a decrease in GSH levels in normal and erbB-transformed fibroblasts, and by an increase in src and raf transformants. Among GSH-related enzymes, only γ-glutamylcysteine synthetase was altered in normal and erbB-transformed NIH/3T3 fibroblasts following PTPase transfection. Therefore, tyrosine phosphorylation could be selectively involved in the regulation of GSH metabolism in normal and oncogene-transformed NIH/3T3 fibroblasts, possibly by a dual-type effect on receptor/oncoprotein-mediated mitogenic signal transduction. However, no relationship was observed between the GSH and PTPase effect on cell growth, either after oncogene transfection or PTPase transfection. Moreover, the changes in GSH metabolism were specifically related to cell lineage. In fact GSH and related enzymes did not change in 32D hematopoietic cells transformed by the same activated erbB oncogene and in those - normal or transformed over-expressing the PTPase: in these cells also, over-expression of the PTPase gene was not accompanied by growth inhibition.

The transcriptional regulator p53 plays an essential role in tumor suppression. Accordingly, it is found mutated, and its activity is reduced, in many human cancers. Recent reports show that some cancers are characterized by a loss of function of wild-type p53, which, in several cases, accumulates in intracellular aggregates. Although the nature of such aggregates is still unclear, recent evidence indicates that the p53 C-terminal and core domains can undergo amyloid aggregation in vitro under mild denaturing conditions, although no information is available on the largely unstructured N-terminal transactivation domain. We therefore decided to investigate the amyloid propensity of the acidic unfolded 1–63 fragment of the transactivation domain, cloned, expressed, and purified from a bacterial strain. Here we show that, when exposed to acidic pH, the 1–63 fragment forms thioflavine T-positive aggregates whose amyloid nature was confirmed by Fourier transform infrared spectroscopy analysis, atomic force microscopy, and x-ray diffraction. These aggregates were shown to be cytotoxic to human SH-SY5Y cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, lactate dehydrogenase release, and caspase-3 activity assays. These results add new significant details to the picture describing the propensity of single domains of p53 to aggregate, further suggesting that, under suitable destabilizing conditions, the whole protein may aggregate into amyloid assemblies in vivo.

In cardiac and skeletal muscle Ca2+ translocation from cytoplasm into sarcoplasmic reticulum (SR) is accomplished by different Ca2+-ATPases whose functioning involves the formation and decomposition of an acylphosphorylated phosphoenzyme intermediate (EP). In this study we found that acylphosphatase, an enzyme well represented in muscular tissues and which actively hydrolyzes EP, had different effects on heart (SERCA2a) and fast twitch skeletal muscle SR Ca2+-ATPase (SERCA1). With physiological acylphosphatase concentrations SERCA2a exhibited a parallel increase in the rates of both ATP hydrolysis and Ca2+ transport; in contrast, SERCA1 appeared to be uncoupled since the stimulation of ATP hydrolysis matched an inhibition of Ca2+ pump. These different effects probably depend on phospholamban, which is associated with SERCA2a but not SERCA1. Consistent with this view, the present study suggests that acylphosphatase-induced stimulation of SERCA2a, in addition to an enhanced EP hydrolysis, may be due to a displacement of phospholamban, thus to a removal of its inhibitory effect.

A number of phosphotyrosine-containing peptides derived from the PDGF receptor phosphorylation sites have been synthesised. The peptides were assayed as substrates of the two isoforms (IF1 and IF2) of the low Mr PTPase. The calculated kcat, Km, and kcat/Km values indicate that only one peptide is best hydrolysed by IF2 (but not IF1), whose catalytic efficiency averages those previously reported for most PTPases (except the Yersinia enzyme). This peptide is the only one containing a couple of no bulky hydrophobic residues at the phosphotyrosine N-side. The determination of the same catalytic parameters in the presence of analogues of the best hydrolysed peptide in which one or both hydrophobic residues were replaced by Asp or Lys residues confirmed the importance of the hydrophobic cluster at the phosphotyrosine N-side for optimal enzymatic hydrolysis. These findings are discussed in the light of the known IF2 X-ray structure.

The interference of low-molecular-weight phosphotyrosine protein phosphatase with the macrophage response to macrophage colony-stimulating factor was investigated. This paper shows that this phosphatase, already known to be involved in platelet-derived growth factor receptor signaling, is physiologically expressed in murine macrophages and dephosphorylates in vitro macrophage colony-stimulating factor receptor molecules immunoprecipitated from macrophage colony-stimulating factor-stimulated macrophages. We obtained the first demonstration that a phosphotyrosine-specific protein phosphatase dephosphorylates the macrophage colony-stimulating factor receptor in vivo and reduces the mitogenic response to macrophage colony-stimulating factor. The data indicate that low-molecular-weight phosphotyrosine protein phosphatase is a negative regulator of macrophage colony-stimulating factor receptor signaling.

Cys21 is an invariant residue in muscle acylphosphatases, but is absent in the erythrocyte isozymes. To assess the importance of this residue in the muscle isozymes for catalytic, structural, and stability properties, two gene mutants have been prepared by oligonucleotide-directed mutagenesis and expressed in Escherichia coli cells; in these mutants, the codon for Cys21 was replaced by those for Ser and Ala, respectively. The two mutant enzymes, purified by immunoaffinity chromatography, showed kinetic and structural properties similar to those of the wild-type recombinant enzyme; however, the specific activity of the two mutants, especially that of the C21A mutant, was lower. The urea and thermal stabilities of the mutant enzymes were reduced with respect to those of the wild-type form, contrary to the susceptibility to inactivation by mercuric ions. The reported data support the possibility that Cys21 is involved in the stabilization of the enzyme active-site conformation.

Low M(r) phosphotyrosine-protein phosphatase belongs to the non-receptor cytosolic phosphotyrosine-protein phosphatase subfamily. It has been demonstrated that this enzyme dephosphorylates receptor tyrosine kinases, namely the epidermal growth factor receptor in vitro and the platelet-derived growth factor receptor in vivo. Low M(r) phosphotyrosine-protein phosphatase is constitutively tyrosine-phosphorylated in NIH/3T3 cells transformed by pp60v-src. The same tyrosine kinase, previously immunoprecipitated, phosphorylates this enzyme in vitro as well. Phosphorylation is enhanced using phosphatase inhibitors and phenylarsine oxide-inactivated phosphatase, consistently with the existence of an auto-dephosphorylation process. Intermolecular dephosphorylation is demonstrated adding the active enzyme in a solution containing the inactivated and previously phosphorylated one. This tyrosine phosphorylation correlates with an increase in catalytic activity. Our results provide evidence of a physiological mechanism of low M(r) phosphotyrosine-protein phosphatase activity regulation.