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Anna Rita Bizzarri1, Simona Santini1, Emilia Coppari1, Monica Bucciantini2, Silvia Di Agostino3, Tohru Yamada4, Craig W Beattie4, Salvatore Cannistraro11Biophysics and Nanoscience Centre, CNISM, Facoltà di Scienze, Università della Tuscia, Viterbo, 2Department of Biochemical Sciences, University of Florence, Florence, 3Molecular Oncogenesis Laboratory, Experimental Oncology Department, Regina Elena Cancer Institute, Rome, Italy; 4Department of Surgical Oncology, University of Illinois College of Medicine, Chicago, IL, USAAbstract: p28 is a 28-amino acid peptide fragment of the cupredoxin azurin derived from Pseudomonas aeruginosa that preferentially penetrates cancerous cells and arrests their proliferation in vitro and in vivo. Its antitumor activity reportedly arises from post-translational stabilization of the tumor suppressor p53 normally downregulated by the binding of several ubiquitin ligases. This would require p28 to specifically bind to p53 to inhibit specific ligases from initiating proteosome-mediated degradation. In this study, atomic force spectroscopy, a nanotechnological approach, was used to investigate the interaction of p28 with full-length p53 and its isolated domains at the single molecule level. Analysis of the unbinding forces and the dissociation rate constant suggest that p28 forms a stable complex with the DNA-binding domain of p53, inhibiting the binding of ubiquitin ligases other than Mdm2 to reduce proteasomal degradation of p53.Keywords: AFS, cancer physics, unbinding forces

α-Synuclein (Syn) is an intrinsically disordered protein, abundant in presynaptic neurons. It is a constituent of the Lewis Body inclusions as amyloid fibrils, in Parkinson's disease patients. It populates an ensemble of conformations and floats between the free random coil and the membrane-bound α-helical species. E46K is a pathogenic mutant of Syn able to accelerate the formation of fibrils. The lysine in position 46 affects several protein structural properties including its interaction with membranes. We have shown that 3,4-dihydroxyphenylacetic acid (DOPAC), a dopamine metabolite, hampers Syn to form fibrils, interfering with the aggregation process and alters the interaction of the protein and its aggregates with membranes. To understand the mechanism of such alteration, we studied the interplay between Syn and E46K, lipid membranes and DOPAC. The ability of DOPAC to displace the proteins bound to membrane was also tested. Our findings provided a dynamic model of interaction able to explain the different effects of DOPAC on lipid binding properties of Syn and E46K, shedding light on the conformational changes induced by the catechol, which may destabilize the protein interactions with membranes. Understanding these mechanisms could have implications for therapeutic strategies targeting Syn aggregation and membrane interactions in neurodegenerative diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

This study introduces a novel multitargeting strategy that combines carbonic anhydrase (CA) activators and cholinesterase (ChE) inhibitors to enhance cognitive functions. A series of tacrine-based derivatives with amine/amino acid moieties were synthesized and evaluated for their dual activity on brain CA isoforms and ChEs (AChE and BChE). Several derivatives, notably compounds 26 , 30 , 34 , and 40 , demonstrated potent CA activation, particularly of hCA II and VII, and strong ChE inhibition with subnanomolar to low nanomolar IC 50 values. In vivo studies using a mouse model of social recognition memory showed that these derivatives significantly improved memory consolidation at doses 10-100 times lower than the reference compounds (either alone or in combination). Molecular modeling and ADMET predictions elucidated the compound binding modes and confirmed favorable pharmacokinetic and safety profiles. The findings suggest that dual modulation of CA and ChE activities is a promising strategy for treating cognitive deficits associated with neurodegenerative and psychiatric disorders.

Accumulation of the pro-inflammatory protein S100A9 has been implicated in neuroinflammatory cascades in neurodegenerative diseases (NDs) such as Alzheimer's disease (AD) and Parkinson's disease (PD). S100A9 co-aggregates with other proteins such as α-synuclein in PD and Aβ in AD, contributing to amyloid plaque formation and neurotoxicity. The amyloidogenic nature of this protein and its role in chronic neuroinflammation suggest that it may play a key role in the pathophysiology of these diseases. Research into molecules targeting S100A9 could be a potential therapeutic strategy to prevent its amyloidogenic self-assembly and to attenuate the neuroinflammatory response in affected brain tissue. This work suggests that bioactive natural molecules, such as those found in the Mediterranean diet, may have the potential to alleviate neuroinflammation associated with the accumulation of proteins such as S100A9 in neurodegenerative diseases. A major component of extra virgin olive oil (EVOO), hydroxytyrosol (HT), with its ability to interact with and modulate S100A9 amyloid self-assembly and expression, offers a compelling approach for the development of novel and effective interventions for the prevention and treatment of ND. The findings highlight the importance of exploring natural compounds, such as HT, as potential therapeutic options for these complex and challenging neurological conditions., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Manuela Leri reports financial support was provided by Umberto Veronesi Foundation. Ludmilla Morozova-Roche reports financial support was provided by Swedish Medical Research Council. Ludmilla Morozova-Roche reports financial support was provided by Strategic research grant funded by the Faculty of Medicine, Umeå University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

We report here the first dual inhibitors of brain carbonic anhydrases (CAs) and monoamine oxidase-B (MAO-B) for the management of Alzheimer's disease. Classical CA inhibitors (CAIs) such as methazolamide prevent amyloid-β-peptide (Aβ)-induced overproduction of reactive oxygen species (ROS) and mitochondrial dysfunction. MAO-B is also implicated in ROS production, cholinergic system disruption, and amyloid plaque formation. In this work, we combined a reversible MAO-B inhibitor of the coumarin and chromone type with benzenesulfonamide fragments as highly effective CAIs. A hit-to-lead optimization led to a significant set of derivatives showing potent low nanomolar inhibition of the target brain CAs ( K I s in the range of 0.1-90.0 nM) and MAO-B (IC 50 in the range of 6.7-32.6 nM). Computational studies were conducted to elucidate the structure-activity relationship and predict ADMET properties. The most effective multitarget compounds totally prevented Aβ-related toxicity, reverted ROS formation, and restored the mitochondrial functionality in an SH-SY5Y cell model surpassing the efficacy of single-target drugs.

The extensive use of agricultural pesticides to improve crop quality and yield significantly increased the risk to the public of exposure to small but repeated doses of pesticides over time through various routes, including skin, by increasing the risk of disease outbreaks. Although much work was conducted to reduce the use of pesticides in agriculture, little attention was paid to prevention, which could reduce the toxicity of pesticide exposure by reducing its impact on human health. Extra virgin olive oil (EVOO), a major component of the Mediterranean diet, exerts numerous health-promoting properties, many of which are attributed to oleuropein aglycone (OleA), the deglycosylated form of oleuropein, which is the main polyphenolic component of EVOO. In this work, three pesticides with different physicochemical and biological properties, namely oxadiazon (OXA), imidacloprid (IMID), and glyphosate (GLYPHO), were compared in terms of metabolic activity, mitochondrial function and epigenetic modulation in an in vitro cellular model of human HaCaT keratinocytes to mimic the pathway of dermal exposure. The potential protective effect of OleA against pesticide-induced cellular toxicity was then evaluated in a cell pre-treatment condition. This study showed that sub-lethal doses of OXA and IMID reduced the metabolic activity and mitochondrial functionality of HaCaT cells by inducing oxidative stress and altering intracellular calcium flux and caused epigenetic modification by reducing histone acetylation H3 and H4. GLYPHO, on the other hand, showed no evidence of cellular toxicity at the doses tested. Pretreatment of cells with OleA was able to protect cells from the damaging effects of the pesticides OXA and IMID by maintaining metabolic activity and mitochondrial function at a controlled level and preventing acetylation reduction, particularly of histone H3. In conclusion, the bioactive properties of OleA reported here could be of great pharmaceutical and health interest, as they could be further studied to design new formulations for the prevention of toxicity from exposure to pesticide use.

The term neuroinflammation defines the reactions of astrocytes and microglia to alterations in homeostasis in the diseased central nervous system (CNS), the exacerbation of which contributes to the neurodegenerative effects of Alzheimer's disease (AD). Local environmental conditions, such as the presence of proinflammatory molecules, mechanical properties of the extracellular matrix (ECM), and local cell-cell interactions, are determinants of glial cell phenotypes. In AD, the load of the cytotoxic/proinflammatory amyloid β (Aβ) peptide is a microenvironmental component increasingly growing in the CNS, imposing time-evolving challenges on resident cells. This study aimed to investigate the temporal and spatial variations of the effects produced by this process on astrocytes and microglia, either directly or by interfering in their interactions. Ex vivo confocal analyses of hippocampal sections from the mouse model TgCRND8 at different ages have shown that overproduction of Aβ peptide induced early and time-persistent disassembly of functional astroglial syncytium and promoted a senile phenotype of reactive microglia, hindering Aβ clearance. In the late stages of the disease, these patterns were altered in the presence of Aβ-plaques, surrounded by typically reactive astrocytes and microglia. Morphofunctional characterization of peri-plaque gliosis revealed a direct contribution of astrocytes in plaque buildup that might result in shielding Aβ-peptide cytotoxicity and, as a side effect, in exacerbating neuroinflammation.

Autophagy plays a key role in cellular, tissue and organismal homeostasis and in the production of the energy load needed at critical times during development and in response to nutrient shortage. Autophagy is generally considered as a pro-survival mechanism, although its deregulation has been linked to non-apoptotic cell death. Autophagy efficiency declines with age, thus contributing to many different pathophysiological conditions, such as cancer, cardiomyopathy, diabetes, liver disease, autoimmune diseases, infections, and neurodegeneration. Accordingly, it has been proposed that the maintenance of a proper autophagic activity contributes to the extension of the lifespan in different organisms. A better understanding of the interplay between autophagy and risk of age-related pathologies is important to propose nutritional and life-style habits favouring disease prevention as well as possible clinical applications aimed at promoting long-term health., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Manuela Leri reports financial support was provided by Umberto Veronesi Foundation., (Copyright © 2023. Published by Elsevier B.V.)

In Alzheimer's disease (AD), microglia, brain resident immune cells, become chronically inflammatory and neurotoxic. In recent years, neuroinflammation has attracted particular interest in the scientific community. The genetic variants of molecules associated with ''microgliopathies'', including the triggering receptor expressed in myeloid cells-2 (TREM2), result in increased risk of developing AD and cognitive decline. We performed a set of in vitro assays using human neuronal (SH-SY5Y) and microglial (BV2 and C13NJ) cell models. Cells were differentially treated with extra virgin olive oil (EVOO) polyphenols, oleuropein aglycone (OleA) and hydroxytyrosol (HT) before adding LPS. We evaluated the protective effects of these EVOO products by a set of biochemical and cell biology assays, including ELISA, MTT, ROS detection, Western blotting and immunofluorescence. Our results provide an integrated understanding of the neuroprotection exerted by polyphenols in terms of: (i) reduction of pro-inflammatory cytokines release (IL-6, IL-8, IP-10 and RANTES); (ii) activation of the TREM2-dependent anti-inflammatory pathway; (iii) enhancement of protective microglial activity favoring the M2 polarization phenotype. Such findings provide new and important insights into the mechanisms by which the dietary olive polyphenols exert beneficial properties against neuroinflammation and neuronal impairment.

Nutrition has relevant consequences for human health and increasing pieces of evidence indicate that medicinal mushrooms have several beneficial effects. One of the main issues in Western countries is represented by the challenges of aging and age-related diseases, such as neurodegenerative disorders. Among these, Parkinson's disease (PD) affects 10 million people worldwide and is associated with α-synuclein misfolding, also found in other pathologies collectively called synucleinopathies. Here, we show that aqueous extracts of two edible mushrooms, Grifola frondosa and Hericium erinaceus , represent a valuable source of β-glucans and exert anti-aging effects in yeast. Their beneficial effects are mediated through the inhibition of the Ras/PKA pathway, with increased expression of heat shock proteins, along with a consistent increase of both mean and maximal lifespans. These fungal extracts also reduce the toxicity of α-synuclein heterologously expressed in yeast cells, resulting in reduced ROS levels, lower α-synuclein membrane localization, and protein aggregation. The neuroprotective activity of G. frondosa extract was also confirmed in a PD model of Drosophila melanogaster . Taken together, our data suggest the use of G. frondosa and H. erinaceus as functional food to prevent aging and age-related disorders, further supporting the neuro-healthy properties of these medicinal mushroom extracts.

Senescence occurs upon critical telomere shortening, or following DNA damage, oncogenic activation, hypoxia and oxidative stress, overall referred to stress-induced premature senescence (SIPS). In response to DNA damage, senescent cells release cytoplasmic chromatin fragments (CCFs), and express an altered secretome, the senescence-associated secretory phenotype (SASP), which contributes to generate a pro-inflammatory and pro-tumoral extracellular milieu. Polyphenols have gained significant attention owing to their anti-inflammatory and anti-tumour activities. Here, we studied the effect of oleuropein aglycone (OLE) and hydroxytyrosol (HT) on DNA damage, CCF appearance and SASP in a model of irradiation-induced senescence. Neonatal human dermal fibroblasts (NHDFs) were γ-irradiated and incubated with OLE, 5 µM and HT, 1 µM. Cell growth and senescence-associated (SA)-β-Gal-staining were used as senescence markers. DNA damage was evaluated by Comet assay, lamin B1 expression, release of CCFs, cyclic GMP-AMP Synthase (cGAS) activation. IL-6, IL-8, MCP-1 and RANTES were measured by ELISA assay. Our results showed that OLE and HT exerted a protective effect on 8 Gy irradiation-induced senescence, preserving lamin B1 expression and reducing cGAS/STING/NFκB-mediated SASP. The ability of OLE and HT to mitigate DNA damage, senescence status and the related SASP in normal cells can be exploited to improve the efficacy and safety of cancer radiotherapy., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

The paper provides a comprehensive and foundational mechanistic framework of hormesis that establishes its centrality in medicine and public health. This hormetic framework is applied to the assessment of olive polyphenols with respect to their capacity to slow the onset and reduce the magnitude of a wide range of age-related disorders and neurodegenerative diseases, including Alzheimer's Disease and Parkinson's Disease. It is proposed that olive polyphenol-induced anti-inflammatory protective effects are mediated in large part via the activation of AMPK and the upregulation of Nrf2 pathway. Consistently, herein we also review the importance of the modulation of Nrf2-related stress responsive vitagenes by olive polyphenols, which at low concentration according to the hormesis theory activates this neuroprotective cascade to preserve brain health and its potential use in the prevention and therapy against aging and age-related cognitive disorders in humans., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The release of monomers from the homotetrameric protein transthyretin (TTR) is the first event of a cascade, eventually leading to sporadic or familial TTR amyloidoses. Thus, ligands able to stabilize TTR and inhibit monomer release are subject of intense scrutiny as potential treatments against these pathologies. Here, we investigated the interaction between TTR and a non-glycated derivative of the main olive polyphenol, oleuropein (OleA), known to interfere with TTR aggregation. We coupled fluorescence studies with molecular docking to investigate the OleA/TTR interaction using wild-type TTR, a monomeric variant, and the L55P cardiotoxic mutant. We characterized a fluorescence band emitted by OleA upon formation of the OleA/TTR complex. Exploiting this signal, we found that a poorly specific non-stoichiometric interaction occurs on the surface of the protein and a more specific stabilizing interaction takes place in the ligand binding pocket of TTR, exhibiting a K D of 3.23 ± 0.32 µM, with two distinct binding sites. OleA interacts with TTR in different modes, stabilizing it and preventing its dissociation into monomers, with subsequent misfolding. This result paves the way to the possible use of OleA to prevent degenerative diseases associated with TTR misfolding.

The interaction between the cell membrane and misfolded protein species plays a crucial role in the development of neurodegeneration. This study was designed to clarify the relationship between plasma membrane composition in terms of the differently linked sialic acid (Sia) content and cell susceptibility to toxic and misfolded Aβ-42 peptides. The sialylation status in different cell lines was investigated by lectin histochemistry and confocal immunofluorescence and then correlated with the different propensities to bind amyloid fibrils and with the relative cell susceptibility to amyloid damage. This study reveals that expressions of Sias α2,3 and α2,6 linked to galactose/N-acetyl-galactosamine, and PolySia are positively correlated with Aβ-42-induced cell toxicity. PolySia shows an early strong interaction with amyloid fibrils, favoring their binding to GM1 ganglioside containing α2,3 galactose-linked Sia and a loss of cell viability. Our findings demonstrate that cell lines with a prevailing plastic neuron-like phenotype and high monoSia and PolySia contents are highly susceptible to amyloid Aβ-42 toxicity. This toxicity may involve a change in neuron metabolism and promote a compensative/protective increase in PolySia, which, in turn, could favor amyloid binding to GM1, thus exacerbating cell dysmetabolism and further amyloid aggregation.

(1) Background: Autophagy, the major cytoplasmic process of substrate turnover, declines with age, contributing to proteostasis decline, accumulation of harmful protein aggregates, damaged mitochondria and to ROS production. Accordingly, abnormalities in the autophagic flux may contribute to many different pathophysiological conditions associated with ageing, including neurodegeneration. Recent data have shown that extra-virgin olive oil (EVOO) polyphenols stimulate cell defenses against plaque-induced neurodegeneration, mainly, through autophagy induction. (2) Methods: We carried out a set of in vitro experiments on SH-SY5Y human neuroblastoma cells exposed to toxic Aβ 1-42 oligomers to investigate the molecular mechanisms involved in autophagy activation by two olive oil polyphenols, oleuropein aglycone (OleA), arising from the hydrolysis of oleuropein (Ole), the main polyphenol found in olive leaves and drupes and its main metabolite, hydroxytyrosol (HT). (3) Results: Our data show that the mixture of the two polyphenols activates synergistically the autophagic flux preventing cell damage by Aβ 1-42 oligomers., in terms of ROS production, and impairment of mitochondria. (4) Conclusion: Our results support the idea that EVOO polyphenols act synergistically in autophagy modulation against neurodegeneration. These data confirm and provide the rationale to consider these molecules, alone or in combination, as promising candidates to contrast ageing-associated neurodegeneration.

Oxidative stress and inflammation triggered by increased oxidative stress are the cause of many chronic diseases. The lack of anti-inflammatory drugs without side-effects has stimulated the search for new active substances. Plant-derived compounds provide new potential anti-inflammatory and antioxidant molecules. Natural products are structurally optimized by evolution to serve particular biological functions, including the regulation of endogenous defense mechanisms and interaction with other organisms. This property explains their relevance for infectious diseases and cancer. Recently, among the various natural substances, polyphenols from extra virgin olive oil (EVOO), an important element of the Mediterranean diet, have aroused growing interest. Extensive studies have shown the potent therapeutic effects of these bioactive molecules against a series of chronic diseases, such as cardiovascular diseases, diabetes, neurodegenerative disorders and cancer. This review begins from the chemical structure, abundance and bioavailability of the main EVOO polyphenols to highlight the effects and the possible molecular mechanism(s) of action of these compounds against inflammation and oxidation, in vitro and in vivo. In addition, the mechanisms of inhibition of molecular signaling pathways activated by oxidative stress by EVOO polyphenols are discussed, together with their possible roles in inflammation-mediated chronic disorders, also taking into account meta-analysis of population studies and clinical trials.

The interplay between α-synuclein and dopamine derivatives is associated with oxidative stress-dependent neurodegeneration in Parkinson's disease (PD). The formation in the dopaminergic neurons of intraneuronal inclusions containing aggregates of α-synuclein is a typical hallmark of PD. Even though the biochemical events underlying the aberrant aggregation of α-synuclein are not completely understood, strong evidence correlates this process with the levels of dopamine metabolites. In vitro, 3,4-dihydroxyphenylacetaldehyde (DOPAL) and the other two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol (DOPET), share the property to inhibit the growth of mature amyloid fibrils of α-synuclein. Although this effect occurs with the formation of differently toxic products, the molecular basis of this inhibition is still unclear. Here, we provide information on the effect of DOPAC on the aggregation properties of α-synuclein and its ability to interact with membranes. DOPAC inhibits α-synuclein aggregation, stabilizing monomer and inducing the formation of dimers and trimers. DOPAC-induced oligomers did not undergo conformational transition in the presence of membranes, and penetrated the cell, where they triggered autophagic processes. Cellular assays showed that DOPAC reduced cytotoxicity and ROS production induced by α-synuclein aggregates. Our findings show that the early radicals resulting from DOPAC autoxidation produced covalent modifications of the protein, which were not by themselves a primary cause of either fibrillation or membrane binding inhibition. These findings are discussed in the light of the potential mechanism of DOPAC protection against the toxicity of α-synuclein aggregates to better understand protein and catecholamine biology and to eventually suggest a scaffold that can help in the design of candidate molecules able to interfere in α-synuclein aggregation.

Polyphenolic compounds in the Mediterranean diet have received increasing attention due to their protective properties in amyloid neurodegenerative and many other diseases. Here, we have demonstrated for the first time that polyphenol oleuropein aglycone (OleA), which is the most abundant compound in olive oil, has multiple potencies for the inhibition of amyloid self-assembly of pro-inflammatory protein S100A9 and the mitigation of the damaging effect of its amyloids on neuroblastoma SH-SY5Y cells. OleA directly interacts with both native and fibrillar S100A9 as shown by intrinsic fluorescence and molecular dynamic simulation. OleA prevents S100A9 amyloid oligomerization as shown using amyloid oligomer-specific antibodies and cross-β-sheet formation detected by circular dichroism. It decreases the length of amyloid fibrils measured by atomic force microscopy (AFM) as well as reduces the effective rate of amyloid growth and the overall amyloid load as derived from the kinetic analysis of amyloid formation. OleA disintegrates already preformed fibrils of S100A9, converting them into nonfibrillar and nontoxic aggregates as revealed by amyloid thioflavin-T dye binding, AFM, and cytotoxicity assays. At the cellular level, OleA targets S100A9 amyloids already at the membranes as shown by immunofluorescence and fluorescence resonance energy transfer, significantly reducing the amyloid accumulation in GM1 ganglioside containing membrane rafts. OleA increases overall cell viability when neuroblastoma cells are subjected to the amyloid load and alleviates amyloid-induced intracellular rise of reactive oxidative species and free Ca 2+ . Since S100A9 is both a pro-inflammatory and amyloidogenic protein, OleA may effectively mitigate the pathological consequences of the S100A9-dependent amyloid-neuroinflammatory cascade as well as provide protection from neurodegeneration, if used within the Mediterranean diet as a potential preventive measure.

Aging and age-related neurodegeneration are among the major challenges in modern medicine because of the progressive increase in the number of elderly in the world population. Nutrition, which has important long-term consequences for health, is an important way to prevent diseases and achieve healthy aging. The beneficial effects of Vigna unguiculata on metabolic disorders have been widely documented. Here, we show that an aqueous extract of V. unguiculata beans delays senescence both in Saccharomyces cerevisiae and Drosophila melanogaster , in a Snf1/AMPK-dependent manner. Consistently, an increased expression of FOXO, SIRT1, NOTCH and heme oxygenase (HO) genes, already known to be required for the longevity extension in D. melanogaster , is also shown. Preventing α-synuclein self-assembly is one of the most promising approaches for the treatment of Parkinson's disease (PD), for which aging is a risk factor. In vitro aggregation of α-synuclein, its toxicity and membrane localization in yeast and neuroblastoma cells are strongly decreased in the presence of bean extract. In a Caenorhabditis elegans model of PD, V. unguiculata extract substantially reduces the number of the age-dependent degeneration of the cephalic dopaminergic neurons. Our findings support the role of V. unguiculata beans as a functional food in age-related disorders.

Parkinson's disease (PD) is a widespread neurodegenerative disorder characterized by the progressive loss of neurons. The accumulation of aggregated forms of the α-Synuclein (Syn) protein is the main cause of neurotoxicity in PD by disrupting cellular homeostasis until neuronal death. Scientific research is constantly looking for natural products as preventive agents against the progression of several neurodisorders due their safety and non-toxic nature. Neuroprotective phytochemicals include Maysin (Mys), the most abundant C-glycosilflavone in corn silk. In this work, the Mys protective role against damage by Syn amyloid aggregates - oligomers and fibrils - was investigated in SH-SY5Y human neuroblastoma cells obtaining novel and interesting information concerning the Mys molecular mechanism of action. Mys showed effectiveness in preventing the typical toxic events induced by Syn amyloid aggregates, i.e. oxidative stress and imbalance of intracellular calcium homeostasis. Mys exhibited a cytoprotective role, especially against Syn oligomers injury, activating an autophagic degradative process, thus playing a key role on several features of amyloid neurotoxicity. Therefore, Mys could be proposed for the first time to the scientific community as an interesting novel natural compound that might allow to develop alternative strategies to prevent the damage of Syn oligomers involved in Parkinson's disease., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Allium roseum is an important medicinal and aromatic plant, specific to the North African flora and a rich source of important nutrients and bioactive molecules including flavonoids and organosulfur compounds whose biological activities and pharmacological properties are well known. In the present study, the inhibition of amyloid beta protein toxicity by the ethanolic extract of this plant is investigated for the first time. Preliminary biochemical analyses identified kæmpferol and luteolin-7-o-glucoside as the more abundant phenolic compounds. The effects of A. roseum extract (ARE) on aggregation and aggregate cytotoxicity of amyloid beta-42 (Aβ42), whose brain aggregates are a hallmark of Alzheimer's disease, were investigated by biophysical (ThT assay, Dynamic light scattering and transmission electron microscopy) and cellular assays (cytotoxicity, aggregate immunolocalization, ROS measurement and intracellular Ca2+ imaging). The biophysical data suggest that ARE affects the structure of the Aβ42 peptide, inhibits its polymerization, and interferes with the path of fibrillogenesis. The data with cultured cells shows that ARE reduces Aß42 aggregate toxicity by inhibiting aggregate binding to the cell membrane and by decreasing both oxidative stress and intracellular Ca2+. Accordingly, ARE could act as a neuroprotective factor against Aβ aggregate toxicity in Alzheimer's disease., Competing Interests: The authors have declared that no competing interests exist.

Garcinoic acid (GA or δ-T3-13'COOH), is a natural vitamin E metabolite that has preliminarily been identified as a modulator of nuclear receptors involved in β-amyloid (Aβ) metabolism and progression of Alzheimer's disease (AD). In this study, we investigated GA's effects on Aβ oligomer formation and deposition. Specifically, we compared them with those of other vitamin E analogs and the soy isoflavone genistein, a natural agonist of peroxisome proliferator-activated receptor γ (PPARγ) that has therapeutic potential for managing AD. GA significantly reduced Aβ aggregation and accumulation in mouse cortical astrocytes. Similarly to genistein, GA up-regulated PPARγ expression and apolipoprotein E (ApoE) efflux in these cells with an efficacy that was comparable with that of its metabolic precursor δ-tocotrienol and higher than those of α-tocopherol metabolites. Unlike for genistein and the other vitamin E compounds, the GA-induced restoration of ApoE efflux was not affected by pharmacological inhibition of PPARγ activity, and specific activation of pregnane X receptor (PXR) was observed together with ApoE and multidrug resistance protein 1 (MDR1) membrane transporter up-regulation in both the mouse astrocytes and brain tissue. These effects of GA were associated with reduced Aβ deposition in the brain of TgCRND8 mice, a transgenic AD model. In conclusion, GA holds potential for preventing Aβ oligomerization and deposition in the brain. The mechanistic aspects of GA's properties appear to be distinct from those of other vitamin E metabolites and of genistein., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest., (© 2020 Marinelli et al.)

Amyloid aggregates have been demonstrated to exert cytotoxic effects in several diseases. It is widely accepted that the complex and fascinating aggregation pathway involves a series of steps during which many heterogeneous intermediates are generated. This process may be greatly potentiated by the presence of amphipathic components of plasma membrane because they may serve as interaction, condensation, and nucleation points. However, there are few data regarding structural alterations induced by the binding between the amyloid fibrils and membrane components and its direct effects on cell integrity. In this study, we found, by 1-anilinonaphthalene 8-sulfonic acid and transmission electron microscopy/fast Fourier transform, that yeast prion Sup35 oligomers showed higher structural uniformity and altered surface properties when grown in the presence of monosialotetrahexosylganglioside, a component of the cell membrane. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and confocal/sensitized Förster resonance energy transfer analyses revealed that these fibrils showed low cytotoxicity and affinity to plasma membrane. Moreover, time-lapse analysis of Sup35 oligomer fibrillation on cells suggested that the amyloid aggregation process per se exerts cytotoxic effects through the interaction of amyloid intermediates with plasma membrane components. These data provide, to our knowledge, new insights to understand the mechanism of amyloid growth and cytotoxicity in the pathogenesis of amyloid diseases., (Copyright © 2020. Published by Elsevier Inc.)

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease in the elderly people. To date, drugs able to reverse the disease are not available; the gold standard is levodopa that only relieves clinical symptoms, yet with severe side effects after prolonged administration. Many efforts are underway to find alternative targets for PD prevention or treatment, the most promising being α-synuclein (Syn). Recently, we reported that oleuropein aglycone (OleA) interferes with amyloid aggregation of Syn both stabilizing its monomeric state and inducing the formation of harmless, off-pathway oligomers. This study is focused at describing the interaction between Syn and hydroxytyrosol (HT), the phenolic moiety and main metabolite of OleA, and the interferences with Syn aggregation by using biophysical and biological techniques. Our results show that HT dose-dependently inhibits Syn aggregation and that covalent and non-covalent binding mediate HT-Syn interaction. HT does not modify the natively unfolded structure of Syn, rather, it stabilizes specific regions of the molecule leading to inhibition of protein fibrillation. Cellular assays showed that HT reduces the toxicity of Syn aggregates. Moreover, Syn aggregates interaction with the cell membrane, an important factor for prion-like properties of Syn on-pathway oligomers, was reduced in cells exposed to Syn aggregates grown in the presence of HT., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The increasing extension in life expectancy of human beings in developed countries is accompanied by a progressively greater rate of degenerative diseases associated with lifestyle and aging, most of which are still waiting for effective, not merely symptomatic, therapies. Accordingly, at present, the recommendations aimed at reducing the prevalence of these conditions in the population are limited to a safer lifestyle including physical/mental exercise, a reduced caloric intake, and a proper diet in a convivial environment. The claimed health benefits of the Mediterranean and Asian diets have been confirmed in many clinical trials and epidemiological surveys. These diets are characterized by several features, including low meat consumption, the intake of oils instead of fats as lipid sources, moderate amounts of red wine, and significant amounts of fresh fruit and vegetables. In particular, the latter have attracted popular and scientific attention for their content, though in reduced amounts, of a number of molecules increasingly investigated for their healthy properties. Among the latter, plant polyphenols have raised remarkable interest in the scientific community; in fact, several clinical trials have confirmed that many health benefits of the Mediterranean/Asian diets can be traced back to the presence of significant amounts of these molecules, even though, in some cases, contradictory results have been reported, which highlights the need for further investigation. In light of the results of these trials, recent research has sought to provide information on the biochemical, molecular, epigenetic, and cell biology modifications by plant polyphenols in cell, organismal, animal, and human models of cancer, metabolic, and neurodegenerative pathologies, notably Alzheimer's and Parkinson disease. The findings reported in the last decade are starting to help to decipher the complex relations between plant polyphenols and cell homeostatic systems including metabolic and redox equilibrium, proteostasis, and the inflammatory response, establishing an increasingly solid molecular basis for the healthy effects of these molecules. Taken together, the data currently available, though still incomplete, are providing a rationale for the possible use of natural polyphenols, or their molecular scaffolds, as nutraceuticals to contrast aging and to combat many associated pathologies., Competing Interests: The authors declare no conflict of interest

Background: L-Homocysteine (Hcy) is a non-proteinogenic α-amino acid synthesized from dietary methionine. In healthy humans, high Hcy levels are a risk factor for cardiovascular diseases, stroke and type 2 diabetes. A recent study reports that Hcy reacts with Cys 10 of transthyretin (TTR), generating a stable covalent adduct. However, to date the effect of S-homocysteinylation on TTR conformational stability remains unknown., Methods: The effect of Hcy on the conformational properties of wt- and L55P-TTR were analysed using a set of biophysical techniques. The cytotoxicity of S-homocysteinylated L55P-TTR was also evaluated in the HL-1 cardiomyocyte cell line, while the effects of the assemblies on kinematic and dynamics properties of cardiac muscle cells were analysed in cardiomyocyte syncytia., Results: We found that Hcy stabilizes tetrameric wt-TTR, while it destabilizes the tetrameric structure of the L55P mutant, promoting the accumulation of self-assembly-prone monomeric species., Conclusions: Our study demonstrated that S-homocysteinylation of the L55P-TTR mutant impairs protein stability, favouring the appearance of toxic monomers. Interestingly, S-homocysteinylation affected only mutant, not wt-TTR. Moreover, we also show that assemblies of S-homocysteinylated L55P-TTR impair cardiomyocytes functional parameters., General Significance: Our study offers new insights on the negative impact of S-homocysteinylation on L55P-TTR stability, whose aggregation is considered the causative agent of a form of early-onset familial amyloid polyneuropathy and cardiomyopathy. Our results suggest that high homocysteine levels are a further risk factor for TTR cardiomyopathy in patients harbouring the L55P-TTR mutation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Andrographolide (AG) was encapsulated in human albumin nanoparticles (AG NPs), and their crossing properties of the blood-brain barrier (BBB), brain distribution, and effects in TgCRND8 mice were evaluated. The development of appropriate NP formulations is mandatory because of the scarce BBB permeability properties of AG. Developed NPs had proper size (mean size: 159.2 ± 4.5 nm), size distribution (PDI nearby 0.12 ± 0.01), and ζ potential (-24.8 ± 1.2 mV), which were not affected by sodium fluorescein (NAF) loading. When AG was loaded to NPs, it slightly affected their size (210.4 ± 3.2 nm) and ζ potential (-20.3 ± 1.5) but not the PDI. Both NAF and AG had a remarkable encapsulation efficiency (more than 99%). The in vitro release of AG from the NPs reached the highest percentage (48%) after 24 h, and the Higuchi's equation was found to be the best fitting model (R 2 = 0.9635). Both AG and AG NPs did not alter the viability of N2a murine neuroblastoma cells when compared with the untreated control cells. In the step-down inhibitory avoidance test, AG NPs administered to TgCRND8 mice significantly improved their performance (P < 0.0001), reaching levels comparable to those displayed by wild-type mice. In the object recognition test, treated and untreated animals showed no deficiencies in exploratory activity, directional movement toward objects, and locomotor activity. No cognitive impairments (discrimination score) were detected in TgCRND8 mice (P < 0.0001) treated with AG NPs. After acute intravenous administration (200 µl), NPs loaded with the probe NAF were detected in the brain parenchyma of TgCRND8 mice. Immunofluorescent analyses evidenced the presence of NPs both in the pE3-Aβ plaque surroundings and inside the pE3-Aβ plaque, indicative of the ability of these NPs to cross the BBB and to penetrate in both undamaged and damaged brain tissues. Furthermore, the immunohistochemical analysis of GFAP-positive astrocytes in the hippocampus of Tg mice evidenced the anti-inflammatory activity of AG when AG NPs were intraperitoneally administered. AG was not effective in counteracting amyloid Aβ aggregation and the resulting toxicity but significantly decreased the oxidative stress levels. In conclusion, AG NPs have extraordinary versatility, nontoxicity, nonimmunogenicity, strong biocompatibility, high biodegradability, and astonishing loading capacity of drug.

Oleuropein aglycone (OleA), the most abundant polyphenol in extra virgin olive oil (EVOO), and Hydroxythyrosol (HT), the OleA main metabolite, have attracted our interest due to their multitarget effects, including the interference with amyloid aggregation path. However, the mechanistic details of their anti-amyloid effect are not known yet. We report here a broad biophysical approach and cell biology techniques that enabled us to characterize the different molecular mechanisms by which OleA and HT modulate the Aβ 1-42 fibrillation, a main histopathological feature of Alzheimer's disease (AD). In particular, OleA prevents the growth of toxic Aβ 1-42 oligomers and blocks their successive growth into mature fibrils following its interaction with the peptide N-terminus, while HT speeds up harmless fibril formation. Our data demonstrate that, by stabilizing oligomers and fibrils, both polyphenols reduce their seeding activity and aggregate/membrane interaction on human neuroblastoma SH-SY5Y cells. These findings highlight the great potential of EVOO polyphenols and offer the possibility to validate and to optimize their use for possible AD prevention and therapy., (Copyright © 2019. Published by Elsevier Ltd.)

Protein misfolding and aggregation are associated with a number of human degenerative diseases. In spite of the enormous research efforts to develop effective strategies aimed at interfering with the pathogenic cascades induced by misfolded/aggregated peptides/proteins, the necessary detailed understanding of the molecular bases of amyloid formation and toxicity is still lacking. To this aim, approaches able to provide a global insight in amyloid-mediated physiological alterations are of importance. In this study, we exploited Fourier transform infrared microspectroscopy, supported by multivariate analysis, to investigate in situ the spectral changes occurring in cultured intact HL-1 cardiomyocytes exposed to wild type (WT) or mutant (L55P) transthyretin (TTR) in native, or amyloid conformation. The presence of extracellular deposits of amyloid aggregates of WT or L55P TTR, respectively, is a key hallmark of two pathological conditions, known as senile systemic amyloidosis and familial amyloid polyneuropathy. We found that the major effects, associated with modifications in lipid properties and in the cell metabolic/phosphorylation status, were observed when natively folded WT or L55P TTR was administered to the cells. The effects induced by aggregates of TTR were milder and in some cases displayed a different timing compared to those elicited by the natively folded protein.

Mounting evidence indicates soluble Aβ 42 oligomers as the most toxic species causing neuronal death which leads to the onset and progression of Alzheimer disease (AD). Recently, it has been found that neurotoxic Aβ 42 oligomers grow from monomeric species or arise following secondary nucleation by preformed mature fibrils. Thus, the use of natural compounds such as polyphenols to hinder the growth or to remodel Aβ 42 fibrils is one of the most promising strategies for AD treatment. In our previous study, we showed that 1, 2, 4-trihydroxynaphthalene-2-O-β-d-glucopyranoside (THNG) inhibits Aβ 42 aggregation during the early steps of the aggregation process, inhibits its conformational change to a β-sheet-rich structure, decreases its polymerization, inhibits its fibrillogenisis and reduces oxidative stress and aggregate cytotoxicity. Here, we used different spectroscopic and cell culture methods to check the effect of THNG on fibrils disaggregation. We showed that THNG binds to mature Aβ 42 fibrils, rearrange their secondary structure, and remodels them into non-amyloid, less toxic, species by inhibiting their interaction with the plasma membrane. Our findings reveal that THNG is a good agent to remodel amyloid fibrils and could be used as a starting molecular scaffold to design new anti-AD drugs., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Many data highlight the benefits of the Mediterranean diet and its main lipid component, extra-virgin olive oil (EVOO). EVOO contains many phenolic compounds that have been found effective against several aging- and lifestyle-related diseases, including neurodegeneration. Oleuropein, a phenolic secoiroid glycoside, is the main polyphenol in the olive oil. It has been reported that the aglycone form of Oleuropein (OleA) interferes in vitro and in vivo with amyloid aggregation of a number of proteins/peptides involved in amyloid, particularly neurodegenerative, diseases avoiding the growth of toxic oligomers and displaying protection against cognitive deterioration., Methods: In this study, we carried out a cellular and biophysical study on the relationships between the effects of OleA on the aggregation and cell interactions of the D76N β2-microglobulin (D76N b2m) variant associated with a familial form of systemic amyloidosis with progressive bowel dysfunction and extensive visceral amyloid deposits., Results: Our results indicate that OleA protection against D76N b2m cytotoxicity results from i) a modification of the conformational and biophysical properties of its amyloid fibrils; ii) a modification of the cell bilayer surface properties of exposed cells., Conclusions: This study reveals that OleA remodels not only D76N b2m aggregates but also the cell membrane interfering with the misfolded proteins-cell membrane association, in most cases an early event triggering amyloid-mediated cytotoxicity., General Significance: The data provided in the present article focus on OleA protection, featuring this polyphenol as a promising plant molecule useful against amyloid diseases., (Copyright © 2018 Elsevier B.V. All rights reserved.)

α-synuclein plays a key role in the pathogenesis of Parkinson's disease (PD); its deposits are found as amyloid fibrils in Lewy bodies and Lewy neurites, the histopathological hallmarks of PD. Amyloid fibrillation is a progressive polymerization path starting from peptide/protein misfolding and proceeding through the transient growth of oligomeric intermediates widely considered as the most toxic species. Consequently, a promising approach of intervention against PD might be preventing α-synuclein build-up, misfolding and aggregation. A possible strategy involves the use of small molecules able to slow down the aggregation process or to alter oligomer conformation favouring the growth of non-pathogenic species. Here, we show that oleuropein aglycone (OleA), the main olive oil polyphenol, exhibits anti-amyloidogenic power in vitro by interacting with, and stabilizing, α-synuclein monomers thus hampering the growth of on-pathway oligomers and favouring the growth of stable and harmless aggregates with no tendency to evolve into other cytotoxic amyloids. We investigated the molecular basis of such interference by both biophysical techniques and limited proteolysis; aggregate morphology was monitored by electron microscopy. We also found that OleA reduces the cytotoxicity of α-synuclein aggregates by hindering their binding to cell membrane components and preventing the resulting oxidative damage to cells.

Presently, misfolding and aggregation of amyloid-β 42 (Aβ 42 ) are considered early events in Alzheimer's disease (AD) pathogenesis. The use of natural products to inhibit the aggregation process and to protect cells from cytotoxicity of early aggregate grown at the onset of the aggregation path is one of the promising strategies against AD. Recently, we have purified a new powerful antioxidant and inhibitor of Aβ 42 aggregation from the leaves of Lawsonia inermis. The new compound was identified as a new Lawsoniaside; 1,2,4-trihydroxynaphthalene-2-O-β-D-glucopyranoside (THNG). Herein, we show that THNG interferes with Aβ 42 aggregation, inhibits its conformational change to a β-sheet-rich structure, decreases its polymerization into large fibrillar species, reduces oxidative stress, and aggregate cytotoxicity. These results indicate that THNG has great potential as a neuroprotective and therapeutic agent against AD. © 2018 BioFactors, 44(3):272-280, 2018., (© 2018 International Union of Biochemistry and Molecular Biology.)

Systemic amyloidosis is caused by misfolding and aggregation of globular proteins in vivo for which effective treatments are urgently needed. Inhibition of protein self-aggregation represents an attractive therapeutic strategy. Studies on the amyloidogenic variant of β 2 -microglobulin, D76N, causing hereditary systemic amyloidosis, have become particularly relevant since fibrils are formed in vitro in physiologically relevant conditions. Here we compare the potency of two previously described inhibitors of wild type β 2 -microglobulin fibrillogenesis, doxycycline and single domain antibodies (nanobodies). The β 2 -microglobulin -binding nanobody, Nb24, more potently inhibits D76N β 2 -microglobulin fibrillogenesis than doxycycline with complete abrogation of fibril formation. In β 2 -microglobulin knock out mice, the D76N β 2 -microglobulin/ Nb24 pre-formed complex, is cleared from the circulation at the same rate as the uncomplexed protein; however, the analysis of tissue distribution reveals that the interaction with the antibody reduces the concentration of the variant protein in the heart but does not modify the tissue distribution of wild type β 2 -microglobulin. These findings strongly support the potential therapeutic use of this antibody in the treatment of systemic amyloidosis.

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 Ca 2+ levels, and Ca 2+ cycling. The altered intracellular Ca 2+ 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., (Copyright © 2016. Published by Elsevier Inc.)

The first genetic variant of β2 -microglobulin (b2M) associated with a familial form of systemic amyloidosis has been recently described. The mutated protein, carrying a substitution of Asp at position 76 with an Asn (D76N b2M), exhibits a strongly enhanced amyloidogenic tendency to aggregate with respect to the wild-type protein. In this study, we characterized the D76N b2M aggregation path and performed an unprecedented analysis of the biochemical mechanisms underlying aggregate cytotoxicity. We showed that, contrarily to what expected from other amyloid studies, early aggregates of the mutant are not the most toxic species, despite their higher surface hydrophobicity. By modulating ganglioside GM1 content in cell membrane or synthetic lipid bilayers, we confirmed the pivotal role of this lipid as aggregate recruiter favouring their cytotoxicity. We finally observed that the aggregates bind to the cell membrane inducing an alteration of its elasticity (with possible functional unbalance and cytotoxicity) in GM1-enriched domains only, thus establishing a link between aggregate-membrane contact and cell damage., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Transthyretin (TTR) is involved in a subset of familial or sporadic amyloid diseases including senile systemic amyloidosis (SSA), familial amyloid polyneuropathy and cardiomyopathy (FAP/FAC) for which no effective therapy has been found yet. These conditions are characterized by extracellular deposits primarily found in the heart parenchyma and in peripheral nerves whose main component are amyloid fibrils, presently considered the main culprits of cell sufferance. The latter are polymeric assemblies grown from misfolded TTR, either wt or carrying one out of many identified mutations. The recent introduction in the clinical practice of synthetic TTR-stabilizing molecules that reduce protein aggregation provides the rationale to search natural effective molecules able to interfere with TTR amyloid aggregation by hindering the appearance of toxic species or by favoring the growth of harmless aggregates. Here we carried out an in depth biophysical and morphological study on the molecular features of the aggregation of wt- and L55P-TTR involved in SSA or FAP/FAC, respectively, and on the interference with fibril aggregation, stability and toxicity to cardiac HL-1 cells to demonstrate the ability of Oleuropein aglycone (OleA), the main phenolic component of the extra virgin olive oil. We describe the molecular basis of such interference and the resulting reduction of TTR amyloid aggregate cytotoxicity. Our data offer the possibility to validate and optimize the use of OleA or its molecular scaffold to rationally design promising drugs against TTR-related pathologies that could enter a clinical experimental phase., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Alterations of the tightly interwoven neuron/astrocyte interactions are frequent traits of aging, but also favor neurodegenerative diseases, such as Alzheimer disease (AD). These alterations reflect impairments of the innate responses to inflammation-related processes, such as β-amyloid (Aβ) burdening. Multidisciplinary studies, spanning from the tissue to the molecular level, are needed to assess how neuron/astrocyte interactions are influenced by aging. Our study addressed this requirement by joining fluorescence-lifetime imaging microscopy/phasor multiphoton analysis with confocal microscopy, implemented with a novel method to separate spectrally overlapped immunofluorescence and Aβ autofluorescence. By comparing data from young control rats, chronically inflamed rats, and old rats, we identified age-specific alterations of neuron/astrocyte interactions in the hippocampus. We found a correlation between Aβ aggregation (+300 and +800% of aggregated Aβ peptide in chronically inflamed and oldvs.control rats, respectively) and fragmentation (clasmatodendrosis) of astrocyte projections (APJs) (+250 and +1300% of APJ fragments in chronically inflamed and oldvs.control rats, respectively). Clasmatodendrosis, in aged rats, associates with impairment of astrocyte-mediated Aβ clearance (-45% of Aβ deposits on APJs, and +33% of Aβ deposits on neurons in oldvs.chronically inflamed rats). Furthermore, APJ fragments colocalize with Aβ deposits and are involved in novel Aβ-mediated adhesions between neurons. These data define the effects of Aβ deposition on astrocyte/neuron interactions as a key topic in AD biology.-Mercatelli, R., Lana, D., Bucciantini, M., Giovannini, M. G., Cerbai, F., Quercioli, F., Zecchi-Orlandini, S., Delfino, G., Wenk, G. L., Nos, D. Clasmatodendrosis and β-amyloidosis in aging hippocampus., (© FASEB.)

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.

This work aims at elucidating the relation between morphological and physicochemical properties of different ataxin-3 (ATX3) aggregates and their cytotoxicity. We investigated a non-pathological ATX3 form (ATX3Q24), a pathological expanded form (ATX3Q55), and an ATX3 variant truncated at residue 291 lacking the polyQ expansion (ATX3/291Δ). Solubility, morphology and hydrophobic exposure of oligomeric aggregates were characterized. Then we monitored the changes in the intracellular Ca(2+) levels and the abnormal Ca(2+) signaling resulting from aggregate interaction with cultured rat cerebellar granule cells. ATX3Q55, ATX3/291Δ and, to a lesser extent, ATX3Q24 oligomers displayed similar morphological and physicochemical features and induced qualitatively comparable time-dependent intracellular Ca(2+) responses. However, only the pre-fibrillar aggregates of expanded ATX3 (the only variant which forms bundles of mature fibrils) triggered a characteristic Ca(2+) response at a later stage that correlated with a larger hydrophobic exposure relative to the two other variants. Cell interaction with early oligomers involved glutamatergic receptors, voltage-gated channels and monosialotetrahexosylganglioside (GM1)-rich membrane domains, whereas cell interaction with more aged ATX3Q55 pre-fibrillar aggregates resulted in membrane disassembly by a mechanism involving only GM1-rich areas. Exposure to ATX3Q55 and ATX3/291Δ aggregates resulted in cell apoptosis, while ATX3Q24 was substantially innocuous. Our findings provide insight into the mechanisms of ATX3 aggregation, aggregate cytotoxicity and calcium level modifications in exposed cerebellar cells., (© 2013.)

The interaction of amyloid aggregates with the cell plasma membrane is currently considered among the basic mechanisms of neuronal dysfunction in amyloid neurodegeneration. We used amyloid oligomers and fibrils grown from the yeast prion Sup35p, responsible for the specific prion trait [PSI(+)], to investigate how membrane lipids modulate fibril interaction with the membranes of cultured H-END cells and cytotoxicity. Sup35p shares no homology with endogenous mammalian polypeptide chains. Thus, the generic toxicity of amyloids and the molecular events underlying cell degeneration can be investigated without interference with analogous polypeptides encoded by the cell genome. Sup35 fibrils bound to the cell membrane without increasing its permeability to Ca(2+). Fibril binding resulted in structural reorganization and aggregation of membrane rafts, with GM1 clustering and alteration of its mobility. Sup35 fibril binding was affected by GM1 or its sialic acid moiety, but not by cholesterol membrane content, with complete inhibition after treatment with fumonisin B1 or neuraminidase. Finally, cell impairment resulted from caspase-8 activation after Fas receptor translocation on fibril binding to the plasma membrane. Our observations suggest that amyloid fibrils induce abnormal accumulation and overstabilization of raft domains in the cell membrane and provide a reasonable, although not unique, mechanistic and molecular explanation for fibril toxicity.

The bacterial redox protein azurin has been shown to be able to enter into cancer cells and induce apoptosis by stabilizing p53. Although the formation of a complex between the two proteins has been demonstrated, little is known about their binding features. We investigated the interaction between the transcription activation domain of p53 (p53(1-63)) and Pseudomonas aeruginosa azurin using fluorescence and phosphorescence spectroscopic techniques. Trp phosphorescence lifetime measurements revealed conformational changes in azurin induced by the interaction with p53(1-63). Acrylamide quenching of Trp phosphorescence also indicated a significant increase in the overall flexibility of azurin upon binding to p53(1-63). We show that azurin binds to the N-terminal region of p53 with a dissociation constant in the 5-10 μM range. No change in the fluorescence and phosphorescence emission of p53(1-63) was detected in the presence of azurin. This result indicated that no Trp residue of p53(1-63) is located in the interaction site with azurin and therefore suggested that the azurin binding site does not overlap that of MDM2, the protein that plays a crucial role in the p53 regulation. The present results may assist in the design of novel cancer treatments based on p53 stabilization by azurin., (Copyright © 2011 Elsevier B.V. All rights reserved.)

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.

The discovery of methods suitable for the conversion in vitro of native proteins into amyloid fibrils has shed light on the molecular basis of amyloidosis and has provided fundamental tools for drug discovery. We have studied the capacity of a small library of tetracycline analogues to modulate the formation or destructuration of β2-microglobulin fibrils. The inhibition of fibrillogenesis of the wild type protein was first established in the presence of 20% trifluoroethanol and confirmed under a more physiologic environment including heparin and collagen. The latter conditions were also used to study the highly amyloidogenic variant, P32G. The NMR analysis showed that doxycycline inhibits β2-microglobulin self-association and stabilizes the native-like species through fast exchange interactions involving specific regions of the protein. Cell viability assays demonstrated that the drug abolishes the natural cytotoxic activity of soluble β2-microglobulin, further strengthening a possible in vivo therapeutic exploitation of this drug. Doxycycline can disassemble preformed fibrils, but the IC(50) is 5-fold higher than that necessary for the inhibition of fibrillogenesis. Fibril destructuration is a dynamic and time-dependent process characterized by the early formation of cytotoxic protein aggregates that, in a few hours, convert into non-toxic insoluble material. The efficacy of doxycycline as a drug against dialysis-related amyloidosis would benefit from the ability of the drug to accumulate just in the skeletal system where amyloid is formed. In these tissues, the doxycycline concentration reaches values several folds higher than those resulting in inhibition of amyloidogenesis and amyloid destructuration in vitro.