The food additive fast green FCF inhibits α-synuclein aggregation, disassembles mature fibrils and protects against amyloid-induced neurotoxicity.

α-Synuclein (α-syn) aggregates into cytotoxic amyloid fibrils, which are recognized as the defining neuropathological feature of Parkinson's disease (PD). Therefore, inhibiting α-syn fibrillogenesis and disrupting the preformed fibrils are both considered attractive strategies to cure PD. We discovered that a safe food additive, fast green FCF, is capable of inhibiting α-synuclein fibrillogenesis and reducing the related cytotoxicity. Thioflavin T fluorescence assays demonstrated that fast green FCF could inhibit the fibrillogenesis α-synuclein. In the presence of 100 µM fast green FCF, amorphous aggregates were formed and observed by atomic force microscopy. Toxicity assays in cell cultures revealed that fast green FCF significantly reduced the cytotoxicity of α-syn. Molecular dynamics simulations revealed the potential mechanism of the interactions between fast green FCF and α-synuclein. Fast green FCF greatly disrupted the α-synuclein pentamer and reduced the ß-sheet content by reducing both nonpolar and polar interactions. Furthermore, two binding sites were identified, named region I (Y39-K45) and region II (H50-Q62). Our data reveal that electrostatic interactions, hydrogen bonds, and π-π interactions synergistically contribute to the binding of fast green FCF to the α-synuclein pentamer. These results indicate that fast green FCF is a candidate prototype for the development of drugs against the aggregation of amyloid fibrils in PD.

Engineering Secretory Amyloids for Remote and Highly Selective Destruction of Metastatic Foci.

Functional amyloids produced in bacteria as nanoscale inclusion bodies are intriguing but poorly explored protein materials with wide therapeutic potential. Since they release functional polypeptides under physiological conditions, these materials can be potentially tailored as mimetic of secretory granules for slow systemic delivery of smart protein drugs. To explore this possibility, bacterial inclusion bodies formed by a self-assembled, tumor-targeted Pseudomonas exotoxin (PE24) are administered subcutaneously in mouse models of human metastatic colorectal cancer, for sustained secretion of tumor-targeted therapeutic nanoparticles. These proteins are functionalized with a peptidic ligand of CXCR4, a chemokine receptor overexpressed in metastatic cancer stem cells that confers high selective cytotoxicity in vitro and in vivo. In the mouse models of human colorectal cancer, time-deferred anticancer activity is detected after the subcutaneous deposition of 500 µg of PE24-based amyloids, which promotes a dramatic arrest of tumor growth in the absence of side toxicity. In addition, long-term prevention of lymphatic, hematogenous, and peritoneal metastases is achieved. These results reveal the biomedical potential and versatility of bacterial inclusion bodies as novel tunable secretory materials usable in delivery, and they also instruct how therapeutic proteins, even with high functional and structural complexity, can be packaged in this convenient format.

Self-Assembly of Artificial Sweetener Aspartame Yields Amyloid-like Cytotoxic Nanostructures.

Recent reports have revealed the intrinsic propensity of single aromatic metabolites to undergo self-assembly and form nanostructures of amyloid nature. Hence, identifying whether aspartame, a universally consumed artificial sweetener, is inherently aggregation prone becomes an important area of investigation. Although the reports on aspartame-linked side effects describe a multitude of metabolic disorders, the mechanistic understanding of such destructive effects is largely mysterious. Since aromaticity, an aggregation-promoting factor, is intrinsic to aspartame's chemistry, it is important to know whether aspartame can undergo self-association and if such a property can predispose any cytotoxicity to biological systems. Our study finds that aspartame molecules, under mimicked physiological conditions, undergo a spontaneous self-assembly process yielding regular ß-sheet-like cytotoxic nanofibrils of amyloid nature. The resultant aspartame fibrils were found to trigger amyloid cross-seeding and become a toxic aggregation trap for globular proteins, Aß peptides, and aromatic metabolites that convert native structures to ß-sheet-like fibrils. Aspartame fibrils were also found to induce hemolysis, causing DNA damage resulting in both apoptosis and necrosis-mediated cell death. Specific spatial arrangement between aspartame molecules is predicted to form a regular amyloid-like architecture with a sticky exterior that is capable of promoting viable H-bonds, electrostatic interactions, and hydrophobic contacts with biomolecules, leading to the onset of protein aggregation and cell death. Results reveal that the aspartame molecule is inherently amyloidogenic, and the self-assembly of aspartame becomes a toxic trap for proteins and cells, exposing the bitter side of such a ubiquitously used artificial sweetener.

Cyclic undecapeptide Cyclosporin A mediated inhibition of amyloid synthesis: Implications in alleviation of amyloid induced neurotoxicity.

Amyloids are highly organized fibril aggregates arise from inappropriately folded form of the protein or polypeptide precursors under both physiological as well as simulated ambience. Amyloid synthesis is a multistep process that involves formation of several metastable intermediates. Among various intermediate species, the as-formed soluble oligomers are extremely toxic to the neuronal cells. In the present study, we evaluated cyclosporine A (CsA), an undecapeptide, for its potential to prevent aggregation of model protein ovalbumin (OVA). In an attempt to elucidate involved operative mechanism, the preliminary studies delineate that CsA affects both primary nucleation as well as other secondary pathways involved in OVA fibrillation process. By its specific interaction with amyloid intermediates, the cyclic peptide CsA seems to regulate the lag phase of the fibrillation process in concentration dependent manner. The present study further suggests that exposure to CsA during lag phase ensues in reversal of OVA fibrillation process. On the contrary, mature OVA fibril remained impervious to the CsA treatment. The cyclic undecapeptide CsA was also found to successfully alleviate amyloid induced toxicity in neuroblastoma cells.

Oleuropein aglycone: A polyphenol with different targets against amyloid toxicity.

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 ß2-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.

Amyloidosis-inducing activity of blood cells in mouse AApoAII amyloidosis.

Mouse senile amyloidosis is a disorder in which apolipoprotein A-II (APOA2) deposits as amyloid fibrils (AApoAII) in many organs. We previously reported that AApoAII amyloidosis can be transmitted by feces, milk, saliva and muscle originating from mice with amyloid deposition. In this study, the ability of blood components to transmit amyloidosis was evaluated in our model system. Blood samples were collected from SAMR1.SAMP1-Apoa2c amyloid-laden or amyloidosis-negative mice. The samples were fractionated into plasma, white blood cell (WBC) and red blood cell (RBC) fractions. Portions of each were further separated into soluble and insoluble fractions. These fractions were then injected into recipient mice to determine amyloidosis-induction activities (AIA). The WBC and RBC fractions from amyloid-laden mice but not from amyloidosis-negative mice induced AApoAII amyloid deposition in the recipients. The AIA of WBC fraction could be attributed to AApoAII amyloid fibrils because amyloid fibril-like materials and APOA2 antiserum-reactive proteins were observed in the insoluble fraction of the blood cells. Unexpectedly, the plasma of AApoAII amyloidosis-negative as well as amyloid-laden mice showed AIA, suggesting the presence of substances in mouse plasma other than AApoAII fibrils that could induce amyloid deposition. These results indicated that AApoAII amyloidosis could be transmitted across tissues and between individuals through blood cells.

Amyloid fibrils from the N-terminal prion protein fragment are infectious.

Recombinant C-terminally truncated prion protein PrP23-144 (which corresponds to the Y145Stop PrP variant associated with a Gerstmann-Sträussler-Scheinker-like prion disease) spontaneously forms amyloid fibrils with a parallel in-register ß-sheet architecture and ß-sheet core mapping to residues ∼112-139. Here we report that mice (both tga20 and wild type) inoculated with a murine (moPrP23-144) version of these fibrils develop clinical prion disease with a 100% attack rate. Remarkably, even though fibrils in the inoculum lack the entire C-terminal domain of PrP, brains of clinically sick mice accumulate longer proteinase K-resistant (PrPres) fragments of ∼17-32 kDa, similar to those observed in classical scrapie strains. Shorter, Gerstmann-Sträussler-Scheinker-like PrPres fragments are also present. The evidence that moPrP23-144 amyloid fibrils generated in the absence of any cofactors are bona fide prions provides a strong support for the protein-only hypothesis of prion diseases in its pure form, arguing against the notion that nonproteinaceous cofactors are obligatory structural components of all infectious prions. Furthermore, our finding that a relatively short ß-sheet core of PrP23-144 fibrils (residues ∼112-139) with a parallel in-register organization of ß-strands is capable of seeding the conversion of full-length prion protein to the infectious form has important implications for the ongoing debate regarding structural aspects of prion protein conversion and molecular architecture of mammalian prions.

Amyloid deposits derived from transthyretin in the ligamentum flavum as related to lumbar spinal canal stenosis.

Amyloidosis is a protein conformational disorder with the distinctive feature of extracellular accumulation of amyloid fibrils that come from different proteins. In the ligamentum flavum of the lumbar spine, amyloid deposits were frequently found in elderly patients with lumbar spinal canal stenosis and were at least partially formed by wild-type transthyretin. However, how amyloid deposits in the ligamentum flavum affect lumbar spinal canal stenosis has remained unclear. In this study, we analyzed clinical, pathologic, and radiologic findings of patients with lumbar spinal canal stenosis who had amyloid deposits in the ligamentum flavum. We studied 95 ligamentum flavum specimens obtained from 56 patients with lumbar spinal canal stenosis and 21 ligamentum flavum specimens obtained from 19 patients with lumbar disk herniation. We evaluated histopathologic findings and clinicoradiologic manifestations, such as thickness of the ligamentum flavum and lumbar spinal segmental instability. We found that all 95 ligamentum flavum specimens resected from patients with lumbar spinal canal stenosis had amyloid deposits, which we classified into two types, transthyretin-positive and transthyretin-negative, and that transthyretin amyloid formation in the ligamentum flavum of patients with lumbar spinal canal stenosis was an age-associated phenomenon. The amount of amyloid in the ligamentum flavum was related to clinical manifestations of lumbar spinal canal stenosis, such as thickness of the ligamentum flavum and lumbar spinal segmental instability, in the patients with lumbar spinal canal stenosis with transthyretin-positive amyloid deposits. To our knowledge, this report is the first to show clinicopathologic correlations in transthyretin amyloid deposits of the ligamentum flavum. In conclusion, transthyretin amyloid deposits in the ligamentum flavum may be related to the pathogenesis of lumbar spinal canal stenosis in elderly patients.

Bilberry anthocyanins neutralize the cytotoxicity of co-chaperonin GroES fibrillation intermediates.

The co-chaperonin GroES (Hsp10) works with chaperonin GroEL (Hsp60) to facilitate the folding reactions of various substrate proteins. Upon forming a specific disordered state in guanidine hydrochloride, GroES is able to self-assemble into amyloid fibrils similar to those observed in various neurodegenerative diseases. GroES therefore is a suitable model system to understand the mechanism of amyloid fibril formation. Here, we determined the cytotoxicity of intermediate GroES species formed during fibrillation. We found that neuronal cell death was provoked by soluble intermediate aggregates of GroES, rather than mature fibrils. The data suggest that amyloid fibril formation and its associated toxicity toward cell might be an inherent property of proteins irrespective of their correlation with specific diseases. Furthermore, with the presence of anthocyanins that are abundant in bilberry, we could inhibit both fibril formation and the toxicity of intermediates. Addition of bilberry anthocyanins dissolved the toxic intermediates and fibrils, and the toxicity of the intermediates was thus neutralized. Our results suggest that anthocyanins may display a general and potent inhibitory effect on the amyloid fibril formation of various conformational disease-causing proteins.

Experimental induction and oral transmission of avian AA amyloidosis in vaccinated white hens.

Avian AA amyloidosis is commonly observed in adult birds afflicted with bacterial infections or chronic inflammatory disorders. Experimental AA amyloidosis in birds can be induced by repeated inflammatory stimulation, such as injection with casein or vaccination with oil-emulsified bacterins. However, the transmission of amyloidosis among avian species has not been studied well to date. In the present study, we confirm the potential induction of avian AA amyloidosis by inoculation of Salmonella enteritidis (SE) vaccine or Mycoplasma gallisepticum vaccine. To determine the transmission of chicken AA amyloidosis among white hens, we induced experimental AA amyloidosis in vaccinated chickens by intravenous or oral administration of chicken AA fibrils. Amyloid deposits were observed in chickens injected with SE and inoculated with chicken AA fibrils intravenously (21/26: 81%) and orally (8/12: 67%). These results suggest that chicken AA amyloidosis can be induced by vaccinations, and may be transmitted among like species by oral administration.

Ameliorative effects of telmisartan on the inflammatory response and impaired spatial memory in a rat model of Alzheimer's disease incorporating additional cerebrovascular disease factors.

Telmisartan, an angiotensin type 1 receptor blocker, is used in the management of hypertension to control blood pressure. In addition, telmisartan has a partial agonistic effect on peroxisome proliferator activated receptor γ (PPARγ). Recently, the effects of telmisartan on spatial memory or the inflammatory response were monitored in a mouse model of Alzheimer's disease (AD). However, to date, no studies have investigated the ameliorative effects of telmisartan on impaired spatial memory and the inflammatory response in an AD animal model incorporating additional cerebrovascular disease factors. In this study, we examined the effect of telmisartan on spatial memory impairment and the inflammatory response in a rat model of AD incorporating additional cerebrovascular disease factors. Rats were subjected to cerebral ischemia and an intracerebroventricular injection of oligomeric or aggregated amyloid-ß (Aß). Oral administration of telmisartan (0.3, 1, 3 mg/kg/d) seven days after ischemia and Aß treatment resulted in better performance in the eight arm radial maze task in a dose-dependent manner. Telmisartan also reduced tumor necrosis factor α mRNA expression in the hippocampal region of rats with impaired spatial memory. These effects of telmisartan were antagonized by GW9662, an antagonist of PPARγ. These results suggest that telmisartan has ameliorative effects on the impairment of spatial memory in a rat model of AD incorporating additional cerebrovascular disease factors via its anti-inflammatory effect.

Correlation between protective immunity to α-synuclein aggregates, oxidative stress and inflammation.

OBJECTIVE: Protein aggregation leading to central amyloid deposition is implicated in Parkinson's disease (PD). During disease progression, inflammation and oxidative stress may well invoke humoral immunity against pathological aggregates of PD-associated α-synuclein. The aim was to investigate any possible concurrence between autoimmune responses to α-synuclein monomers, oligomers or fibrils with oxidative stress and inflammation. METHODS: The formation of α-synuclein amyloid species was assessed by thioflavin-T assay and atomic force microscopy was employed to confirm their morphology. Serum autoantibody titers to α-synuclein conformations were determined by ELISA. Enzyme activity and concentrations of oxidative stress/inflammatory indicators were evaluated by enzyme and ELISA protocols. RESULTS: In PD patient sera, a differential increase in autoantibody titers to α-synuclein monomers, toxic oligomers or fibrils was associated with boosted levels of the pro-inflammatory cytokine interleukin-6 and tumour necrosis factor-α, but a decrease in interferon-γ concentration. In addition, levels of malondialdehyde were elevated whilst those of glutathione were reduced along with decrements in the activity of the antioxidants: superoxide dismutase, catalase and glutathione transferase. CONCLUSIONS: It is hypothesized that the generation of α-synuclein amyloid aggregates allied with oxidative stress and inflammatory reactions may invoke humoral immunity protecting against dopaminergic neuronal death. Hence, humoral immunity is a common integrative factor throughout PD progression which is directed towards prevention of further neurodegeneration, so potential treatment strategies should attempt to maintain PD patient immune status.

Intracerebral inoculation of pathological α-synuclein initiates a rapidly progressive neurodegenerative α-synucleinopathy in mice.

The accumulation of misfolded proteins is a fundamental pathogenic process in neurodegenerative diseases. However, the factors that trigger aggregation of α-Synuclein (α-Syn), the principal component of the intraneuronal inclusions known as Lewy bodies (LBs), and Lewy neurites (LNs), which characterize Parkinson's disease (PD) and dementia with LBs (DLB), are poorly understood. We show here that in young asymptomatic α-Syn transgenic (Tg) mice, intracerebral injections of brain homogenates derived from older Tg mice exhibiting α-Syn pathology accelerate both the formation of intracellular LB/LN-like inclusions and the onset of neurological symptoms in recipient animals. Pathological α-Syn propagated along major central nervous system (CNS) pathways to regions far beyond injection sites and reduced survival with a highly reproducible interval from injection to death in inoculated animals. Importantly, inoculation with α-Syn amyloid fibrils assembled from recombinant human α-Syn induced identical consequences. Furthermore, we show for the first time that synthetic α-Syn fibrils are wholly sufficient to initiate PD-like LBs/LNs and to transmit disease in vivo. Thus, our data point to a prion-like cascade in synucleinopathies whereby cell-cell transmission and propagation of misfolded α-Syn underlie the CNS spread of LBs/LNs. These findings open up new avenues for understanding the progression of PD and for developing novel therapeutics.

Shear flow induced changes in apolipoprotein C-II conformation and amyloid fibril formation.

The misfolding and self-assembly of proteins into amyloid fibrils that occur in several debilitating diseases are affected by a variety of environmental factors, including mechanical factors associated with shear flow. We examined the effects of shear flow on amyloid fibril formation by human apolipoprotein C-II (apoC-II). Shear fields (150, 300, and 500 s(-1)) accelerated the rate of apoC-II fibril formation (1 mg/mL) approximately 5-10-fold. Fibrils produced at shear rates of 150 and 300 s(-1) were similar to the twisted ribbon fibrils formed in the absence of shear, while at 500 s(-1), tangled ropelike structures were observed. The mechanism of the shear-induced acceleration of amyloid fibril formation was investigated at low apoC-II concentrations (50 µg/mL) where fibril formation does not occur. Circular dichroism and tryptophan fluorescence indicated that shear induced an irreversible change in apoC-II secondary structure. Fluorescence resonance energy transfer experiments using the single tryptophan residue in apoC-II as the donor and covalently attached acceptors showed that shear flow increased the distance between the donor and acceptor molecules. Shear-induced higher-order oligomeric species were identified by sedimentation velocity experiments using fluorescence detection, while fibril seeding experiments showed that species formed during shear flow are on the fibril formation pathway. These studies suggest that physiological shear flow conditions and conditions experienced during protein manufacturing can exert significant effects on protein conformation, leading to protein misfolding, aggregation, and amyloid fibril formation.

Hydralazine modifies Aß fibril formation and prevents modification by lipids in vitro.

Lipid oxidative damage and amyloid ß (Aß) misfolding contribute to Alzheimer's disease (AD) pathology. Thus, the prevention of oxidative damage and Aß misfolding are attractive targets for drug discovery. At present, no AD drugs approved by the Food and Drug Administration (FDA) prevent or halt disease progression. Hydralazine, a smooth muscle relaxant, is a potential drug candidate for AD drug therapy as it reduces Aß production and prevents oxidative damage via its antioxidant hydrazide group. We evaluated the efficacy of hydralazine, and related hydrazides, in reducing (1) Aß misfolding and (2) Aß protein modification by the reactive lipid 4-hydroxy-2-nonenal (HNE) using transmission electron microscopy and Western blotting. While hydralazine did not prevent Aß aggregation as measured using the protease protection assay, there were more oligomeric species observed by electron microscopy. Hydralazine prevented lipid modification of Aß, and Aß was used as a proxy for classes of proteins which either misfold or are modified by HNE. All of the other hydrazides prevented lipid modification of Aß and also did not prevent Aß aggregation. Surprisingly, a few of the compounds, carbazochrome and niclosamide, appeared to augment Aß formation. Thus, hydrazides reduced lipid oxidative damage, and hydralazine additionally reduced Aß misfolding. While hydralazine would require specific chemical modifications for use as an AD therapeutic itself (to improve blood brain barrier permeability, reduce vasoactive side effects, and optimization for amyloid inhibition), this study suggests its potential merit for further AD drug development.

Regulation of ß cleavage of amyloid precursor protein.

Alzheimer's disease ranks the first cause for senile dementia. The amyloid cascade is proposed to contribute to the pathogenesis of this disease. In this cascade, amyloid ß peptide (Aß) is produced through a sequential cleavage of amyloid precursor protein (APP) by ß and γ secretases, while its cleavage by α secretase precludes Aß production and generates neurotrophic sAPPα. Thus, enhancing α secretase activity or suppressing ß and γ cleavage may reduce Aß formation and ameliorate the pathological process of the disease. Several regulatory mechanisms of APP cleavage have been established. The present review mainly summarizes the signaling pathways pertinent to the regulation of APP ß cleavage.

Retardation of Abeta fibril formation by phospholipid vesicles depends on membrane phase behavior.

An increasing amount of evidence suggests that in several amyloid diseases, the fibril formation in vivo and the mechanism of toxicity both involve membrane interactions. We have studied Alzheimer's disease related amyloid beta peptide (Abeta). Recombinant Abeta(M1-40) and Abeta(M1-42) produced in Escherichia coli, allows us to carry out large scale kinetics assays with good statistics. The amyloid formation process is followed in means of thioflavin T fluorescence at relatively low (down to 380 nM) peptide concentration approaching the physiological range. The lipid membranes are introduced in the system as large and small unilamellar vesicles. The aggregation lagtime increases in the presence of lipid vesicles for all situations investigated and the phase behavior of the membrane in the vesicles has a large effect on the aggregation kinetics. By comparing vesicles with different membrane phase behavior we see that the solid gel phase dipalmitoylphosphatidylcholine bilayers cause the largest retardation of Abeta fibril formation. The membrane-induced retardation reaches saturation and is present when the vesicles are added during the lag time up to the nucleation point. No significant difference is detected in lag time when increasing amount of negative charge is incorporated into the membrane.