Biochemical and Electrophysiological Modification of Amyloid Transthyretin on Cardiomyocytes.

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.

Nutraceutical Properties of Olive Oil Polyphenols. An Itinerary from Cultured Cells through Animal Models to Humans.

The increasing interest in the Mediterranean diet hinges on its healthy and anti-ageing properties. The composition of fatty acids, vitamins and polyphenols in olive oil, a key component of this diet, is considered a key feature of its healthy properties. Therefore, it is of significance that the Rod of Asclepius lying on a world map surrounded by olive tree branches has been chosen by the World Health Organization as a symbol of both peace and well-being. This review travels through most of the current and past research, recapitulating the biochemical and physiological correlations of the beneficial properties of olive tree (Olea europaea) polyphenols and their derivatives found in olive oil. The factors influencing the content and beneficial properties of olive oil polyphenols will also be taken into account together with their bioavailability. Finally, the data on the clinical and epidemiological relevance of olive oil and its polyphenols for longevity and against age- and lifestyle-associated pathologies such as cancer, cardiovascular, metabolic and neurodegenerative diseases are reviewed.

Oleuropein aglycone induces autophagy via the AMPK/mTOR signalling pathway: a mechanistic insight.

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 (Aß) deposition. We found that OLE triggers autophagy in cultured cells through the Ca2+-CAMKKß-AMPK axis. In particular, in these cells OLE induces a rapid release of Ca2+ from the SR stores which, in turn, activates CAMKKß, 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.

Olive Oil Phenols as Promising Multi-targeting Agents Against Alzheimer's Disease.

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 (Aß) 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.

Oleuropein Aglycone: A Possible Drug against Degenerative Conditions. In Vivo Evidence of its Effectiveness 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-ß protein (Aß) 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.

Beneficial properties of natural phenols: highlight on protection against pathological conditions associated with amyloid aggregation.

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.

Employing Alzheimer disease animal models for translational research: focus on dietary components.

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-ß deposition. METHODS: TgCRND8 mice (3.5 months old), expressing the mutant KM670/671NL+V717F h-ßAPP695 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.

The polyphenol oleuropein aglycone protects TgCRND8 mice against Aß plaque pathology.

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.

Oleuropein aglycone protects transgenic C. elegans strains expressing Aß42 by reducing plaque load and motor deficit.

The presence of amyloid aggregates of the 42 amino acid peptide of amyloid beta (Aß42) in the brain is the characteristic feature of Alzheimer's disease (AD). Amyloid beta (Aß deposition is also found in muscle fibers of individuals affected by inclusion body myositis (sIBM), a rare muscular degenerative disease affecting people over 50. Both conditions are presently lacking an effective therapeutic treatment. There is increasing evidence to suggest that natural polyphenols may prevent the formation of toxic amyloid aggregates; this applies also to oleuropein aglycone (OLE), the most abundant polyphenol in extra virgin olive oil, previously shown to hinder amylin and Aß aggregation. Here we evaluated the ability of OLE to interfere with Aß proteotoxicity in vivo by using the transgenic CL2006 and CL4176 strains of Caenorhabditis elegans, simplified models of AD and of sIBM, which express human Aß in the cytoplasm of body wall muscle cells. OLE-fed CL2006 worms displayed reduced Aß plaque deposition, less abundant toxic Aß oligomers, remarkably decreased paralysis and increased lifespan with respect to untreated animals. A protective effect was also observed in CL4176 worms but only when OLE was administered before the induction of the Aß transgene expression. These effects were specific, dose-related, and not mediated by the known polyphenolic anti-oxidant activity, suggesting that, in this model organism, OLE interferes with the Aß aggregation skipping the appearance of toxic species, as already shown in vitro for Aß42.

Mild exposure of RIN-5F ß-cells to human islet amyloid polypeptide aggregates upregulates antioxidant enzymes via NADPH oxidase-RAGE: an hormetic stimulus.

The presence of amyloid aggregates of human islet amyloid polypeptide (hIAPP), a hallmark of type 2 diabetes, contributes to pancreatic ß-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 ß-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 ß-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.

Aß(1-42) aggregates into non-toxic amyloid assemblies in the presence of the natural polyphenol oleuropein aglycon.

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 Aß(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 Aß(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 Aß(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.

Oleuropein aglycon prevents cytotoxic amyloid aggregation of human amylin.

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.

The (1-63) region of the p53 transactivation domain aggregates in vitro into cytotoxic amyloid assemblies.

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.

The low-molecular-weight phosphotyrosine phosphatase is a negative regulator of FcgammaRIIA-mediated cell activation.

Activation of human platelets by cross-linking of the low-affinity receptor for immunoglobulin G (FcgammaRIIA) 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 FcgammaRIIA cross-linking. In vitro, LMW-PTP was found to efficiently dephosphorylate activated FcgammaRIIA and LAT, but not Syk or phospholipase Cgamma2. In the megakaryocytic cell line DAMI, antibody-induced phosphorylation of FcgammaRIIA was rapid and transient. The late dephosphorylation of FcgammaRIIA 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 FcgammaRIIA, and caused a more rapid dephosphorylation. In addition, overexpression of LMW-PTP inhibited activation of Syk downstream of FcgammaRIIA and reduced intracellular Ca(2+) mobilization. These results demonstrate that LMW-PTP is responsible for FcgammaRIIA dephosphorylation, and is implicated in the down-regulation of cell activation mediated by this ITAM-bearing immunoreceptor.

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.

Patterns of cell death triggered in two different cell lines by HypF-N prefibrillar aggregates.

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.

LMW-PTP associates and dephosphorylates STAT5 interacting with its C-terminal domain.

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.