Inhibition of HEWL fibril formation by taxifolin: Mechanism of action.

Among therapeutic approaches for amyloid-related diseases, attention has recently turned to the use of natural products as effective anti-aggregation compounds. Although a wealth of in vitro and in vivo evidence indicates some common inhibitory activity of these compounds, they don't generally suggest the same mechanism of action. Here, we show that taxifolin, a ubiquitous bioactive constituent of foods and herbs, inhibits formation of HEWL amyloid fibrils and their related toxicity by causing formation of very large globular, chain-like aggregates. A range of amyloid-specific techniques were employed to characterize this process. We found that taxifolin exerts its effect by binding to HEWL prefibrillar species, rather than by stabilizing the molecule in its native-like state. Furthermore, it's binding results in diverting the amyloid pathway toward formation of very large globular, chain-like aggregates with low ß-sheet content and reduced solvent-exposed hydrophobic patches. ThT fluorescence measurements show that the binding capacity of taxifolin is significantly reduced, upon generation of large protofibrillar aggregates at the end of growth phase. We believe these results may help design promising inhibitors of protein aggregation for amyloid-related diseases.

Structural evidence for native state stabilization of a conformationally labile amyloidogenic transthyretin variant by fibrillogenesis inhibitors.

Several classes of chemicals are able to bind to the thyroxine binding sites of transthyretin (TTR), stabilizing its native state and inhibiting in vitro the amyloidogenic process. The amyloidogenic I84S TTR variant undergoes a large conformational change at moderately acidic pH. Structural evidence has been obtained by X-ray analysis for the native state stabilization of I84S TTR by two chemically distinct fibrillogenesis inhibitors. In fact, they fully prevent the acidic pH-induced protein conformational change as a result of a long-range stabilizing effect. This study provides further support to the therapeutic strategy based on the use of TTR stabilizers as anti-amyloidogenic drugs.

Vitamins K interact with N-terminus α-synuclein and modulate the protein fibrillization in vitro. Exploring the interaction between quinones and α-synuclein.

In the last decades, a series of compounds, including quinones and polyphenols, has been described as having anti-fibrillogenic action on α-synuclein (α-syn) whose aggregation is associated to the pathogenesis of Parkinson's disease (PD). Most of these molecules act as promiscuous anti-amyloidogenic agents, interacting with the diverse amyloidogenic proteins (mostly unfolded) through non-specific hydrophobic interactions. Herein we investigated the effect of the vitamins K (phylloquinone, menaquinone and menadione), which are 1,4-naphthoquinone (1,4-NQ) derivatives, on α-syn aggregation, comparing them with other anti-fibrillogenic molecules such as quinones, polyphenols and lipophilic vitamins. Vitamins K delayed α-syn fibrillization in substoichiometric concentrations, leading to the formation of short, sheared fibrils and amorphous aggregates, which are less prone to produce leakage of synthetic vesicles. In seeding conditions, menadione and 1,4-NQ significantly inhibited fibrils elongation, which could be explained by their ability to destabilize preformed fibrils of α-syn. Bidimensional NMR experiments indicate that a specific site at the N-terminal α-syn (Gly31/Lys32) is involved in the interaction with vitamins K, which is corroborated by previous studies suggesting that Lys is a key residue in the interaction with quinones. Together, our data suggest that 1,4-NQ, recently showed up by our group as a potential scaffold for designing new monoamine oxidase inhibitors, is also capable to modulate α-syn fibrillization in vitro.

Naphthoquinone-tyrptophan reduces neurotoxic Aß*56 levels and improves cognition in Alzheimer's disease animal model.

An increasing body of evidence indicates a role for oligomers of the amyloid-ß peptide (Aß) in the neurotoxicity of this peptide and the pathology of Alzheimer's disease (AD). Several neurotoxic oligomeric forms of Aß have been noted ranging from the larger Amyloid ß-Derived Diffusible Ligands (ADDLs) to smaller trimers and dimers of Aß. More recently a dodecameric form of Aß with a 56 kDa molecular weight, denoted Aß*56, was shown to cause memory impairment in AD model mice. Here, we present for the first time a potential therapeutic strategy for AD that targets the early stages in the formation of neurotoxic Aß*56 oligomers using a modified quinone-Tryptophan small molecule N-(3-chloro-1,4-dihydro-1,4-dioxo-2-naphthalenyl)-L-Tryptophan (Cl-NQTrp). Using NMR spectroscopy we show that this compound binds the aromatic recognition core of Aß and prevents the formation of oligomers. We assessed the effect of Cl-NQTrp in vivo in transgenic flies expressing Aß(1-42) in their nervous system. When these flies were fed with Cl-NQTrp a marked alleviation of their Aß-engendered reduced life span and defective locomotion was observed. Finally, intraperitoneal injection of Cl-NQTrp into an aggressive AD mouse model reduced the level of the Aß*56 species in their brain and reversed their cognitive defects. Further experiments should assess whether this is a direct effect of the drug in the brain or an indirect peripheral effect. This is the first demonstration that targeted reduction of Aß*56 results in amelioration of AD symptoms. This second generation of tryptophan-modified naphthoquinones could therefore serve as potent disease modifying therapeutic for AD.

Brain insulin resistance accelerates Aß fibrillogenesis by inducing GM1 ganglioside clustering in the presynaptic membranes.

Type 2 diabetes mellitus is thought to be a significant risk factor for Alzheimer's disease. Insulin resistance also affects the central nervous system by regulating key processes, such as neuronal survival and longevity, learning and memory. However, the mechanisms underlying these effects remain uncertain. To investigate whether insulin resistance is associated with the assembly of amyloid ß-protein (Aß) at the cell surface of neurons, we inhibited insulin-signalling pathways of primary neurons. The treatments of insulin receptor (IR)-knockdown and a phosphatidylinositol 3-kinase inhibitor (LY294002), but not an extracellular signal-regulated kinase inhibitor, induced an increase in GM1 ganglioside (GM1) levels in detergent-resistant membrane microdomains of the neurons. The aged db/db mouse brain exhibited reduction in IR expression and phosphorylation of Akt, which later induced an increase in the high-density GM1-clusters on synaptosomes. Neurons treated with IR knockdown or LY294002, and synaptosomes of the aged db/db mouse brains markedly accelerated an assembly of Aßs. These results suggest that ageing and peripheral insulin resistance induce brain insulin resistance, which accelerates the assembly of Aßs by increasing and clustering of GM1 in detergent-resistant membrane microdomains of neuronal membranes.

Amyloid-ß induced toxicity involves ganglioside expression and is sensitive to GM1 neuroprotective action.

The effect of Aß25-35 peptide, in its fibrillar and non-fibrillar forms, on ganglioside expression in organotypic hippocampal slice cultures was investigated. Gangliosides were endogenously labeled with D-[1-C(14)] galactose and results showed that Aß25-35 affected ganglioside expression, depending on the peptide aggregation state, that is, fibrillar Aß25-35 caused an increase in GM3 labeling and a reduction in GD1b labeling, whereas the non-fibrillar form was able to enhance GM1 expression. Interestingly, GM1 exhibited a neuroprotective effect in this organotypic model, since pre-treatment of the hippocampal slices with GM1 10 µM was able to prevent the toxicity triggered by the fibrillar Aß25-35, when measured by propidium iodide uptake protocol. With the purpose of further investigating a possible mechanism of action, we analyzed the effect of GM1 treatment (1, 6, 12 and 24h) upon the Aß-induced alterations on GSK3ß dephosphorylation/activation state. Results demonstrated an important effect after 24-h incubation, with GM1 preventing the Aß-induced dephosphorylation (activation) of GSK3ß, a signaling pathway involved in apoptosis triggering and neuronal death in models of Alzheimer's disease. Taken together, present results provide a new and important support for ganglioside participation in development of Alzheimer's disease experimental models and suggest a protective role for GM1 in Aß-induced toxicity. This may be useful for designing new therapeutic strategies for Alzheimer's treatment.

Targeting tau protein in Alzheimer's disease.

Alzheimer's disease (AD) is characterized histopathologically by numerous neurons with neurofibrillary tangles and neuritic (senile) amyloid-beta (Abeta) plaques, and clinically by progressive dementia. Although Abeta is the primary trigger of AD according to the amyloid cascade hypothesis, neurofibrillary degeneration of abnormally hyperphosphorylated tau is apparently required for the clinical expression of this disease. Furthermore, while approximately 30% of normal aged individuals have as much compact plaque burden in the neocortex as is seen in typical cases of AD, in several tauopathies, such as cortical basal degeneration and Pick's disease, neurofibrillary degeneration of abnormally hyperphosphorylated tau in the absence of Abeta plaques is associated with dementia. To date, all AD clinical trials based on Abeta as a therapeutic target have failed. In addition to the clinical pathological correlation of neurofibrillary degeneration with dementia in AD and related tauopathies, increasing evidence from in vitro and in vivo studies in experimental animal models provides a compelling case for this lesion as a promising therapeutic target. A number of rational approaches to inhibiting neurofibrillary degeneration include inhibition of one or more tau protein kinases, such as glycogen synthase kinase-3beta and cyclin-dependent protein kinase 5, activation of the major tau phosphatase protein phosphatase-2A, elevation of beta-N-acetylglucosamine modification of tau through inhibition of beta-N-acetylglucosaminidase or increase in brain glucose uptake, and promotion of the clearance of the abnormally hyperphosphorylated tau by autophagy or the ubiquitin proteasome system.

Amyloid ß-protein aggregation produces highly reproducible kinetic data and occurs by a two-phase process.

Protein aggregation can lead to major disturbances of cellular processes and is associated with several diseases. We report kinetic and equilibrium data by ThT fluorescence and enzyme-linked immunosorbent assay of sufficient quality and reproducibility to form a basis for mechanistic understanding of amyloid ß-peptide (Aß) fibril formation. Starting from monomeric peptide in a pure buffer system without cosolvents, we find that the kinetics of Aß aggregation vary strongly with peptide concentration in a highly predictable manner. The free Aß concentration in equilibrium with fibrils was found to vary with total peptide concentration in a manner expected for a two-phase system. The free versus total Aß concentration was linear up to ca. 0.2 µM, after which free Aß decreased with total Aß toward an asymptotic value. Our results imply that Aß fibril formation arises from a sequence of events in a highly predictable manner.

Inhibition of tau fibrillization by oleocanthal via reaction with the amino groups of tau.

Tau is a microtubule-associated protein that promotes microtubule assembly and stability. In Alzheimer's disease and related tauopathies, tau fibrillizes and aggregates into neurofibrillary tangles. Recently, oleocanthal isolated from extra virgin olive oil was found to display non-steroidal anti-inflammatory activity similar to ibuprofen. As our unpublished data indicates an inhibitory effect of oleocanthal on amyloid beta peptide fibrillization, we reasoned that it might inhibit tau fibrillization as well. Herein, we demonstrate that oleocanthal abrogates fibrillization of tau by locking tau into the naturally unfolded state. Using PHF6 consisting of the amino acid residues VQIVYK, a hexapeptide within the third repeat of tau that is essential for fibrillization, we show that oleocanthal forms an adduct with the lysine via initial Schiff base formation. Structure and function studies demonstrate that the two aldehyde groups of oleocanthal are required for the inhibitory activity. These two aldehyde groups show certain specificity when titrated with free lysine and oleocanthal does not significantly affect the normal function of tau. These findings provide a potential scheme for the development of novel therapies for neurodegenerative tauopathies.

Anti-aggregation and fibril-destabilizing effects of sex hormones on alpha-synuclein fibrils in vitro.

The alpha-synuclein aggregation in the brain is the hallmark of Lewy body diseases, including Parkinson's disease and dementia with Lewy bodies, and multiple system atrophy. Some epidemiological studies have revealed that estrogen therapy reduces the risk of Parkinson's disease in females. We examined the effects of estriol, estradiol, estrone, androstenedione, and testosterone on the formation and destabilization of alpha-synuclein fibrils at pH 7.5 and 37 degrees C in vitro, using fluorescence spectroscopy with thioflavin S and electron microscopy. These sex hormones, especially estriol, significantly exert anti-aggregation and fibril-destabilizing effects; and hence, could be valuable preventive and therapeutic agents for alpha-synucleinopathies.

Inhibition of Alzheimer's amyloid toxicity with a tricyclic pyrone molecule in vitro and in vivo.

Small beta-amyloid (Abeta) 1-42 aggregates are toxic to neurons and may be the primary toxic species in Alzheimer's disease (AD). Methods to reduce the level of Abeta, prevent Abeta aggregation, and eliminate existing Abeta aggregates have been proposed for treatment of AD. A tricyclic pyrone named CP2 is found to prevent cell death associated with Abeta oligomers. We studied the possible mechanisms of neuroprotection by CP2. Surface plasmon resonance spectroscopy shows a direct binding of CP2 with Abeta42 oligomer. Circular dichroism spectroscopy reveals monomeric Abeta42 peptide remains as a random coil/alpha-helix structure in the presence of CP2 over 48 h. Atomic force microscopy studies show CP2 exhibits similar ability to inhibit Abeta42 aggregation as that of Congo red and curcumin. Atomic force microscopy closed-fluid cell study demonstrates that CP2 disaggregates Abeta42 oligomers and protofibrils. CP2 also blocks Abeta fibrillations using a protein quantification method. Treatment of 5x familial Alzheimer's disease mice, a robust Abeta42-producing animal model of AD, with a 2-week course of CP2 resulted in 40% and 50% decreases in non-fibrillar and fibrillar Abeta species, respectively. Our results suggest that CP2 might be beneficial to AD patients by preventing Abeta aggregation and disaggregating existing Abeta oligomers and protofibrils.

Preventing beta-amyloid fibrillization and deposition: beta-sheet breakers and pathological chaperone inhibitors.

Central to the pathogenesis of Alzheimer's disease (AD) is the conversion of normal, soluble beta-amyloid (sAbeta) to oligomeric, fibrillar Abeta. This process of conformational conversion can be influenced by interactions with other proteins that can stabilize the disease-associated state; these proteins have been termed 'pathological chaperones'. In a number of AD models, intervention that block soluble Abeta aggregation, including beta-sheet breakers, and compounds that block interactions with pathological chaperones, have been shown to be highly effective. When combined with early pathology detection, these therapeutic strategies hold great promise as effective and relatively toxicity free methods of preventing AD related pathology.

Sensitive fluorescence polarization technique for rapid screening of alpha-synuclein oligomerization/fibrillization inhibitors.

Parkinson's disease (PD) is characterized by the accumulation of fibrillar alpha-synuclein (alpha-Syn) inclusions known as Lewy bodies (LBs) and Lewy neurites. Mutations in the alpha-Syn gene or extra copies thereof cause familial PD or dementia with LBs (DLB) in rare kindreds, but abnormal accumulations of wildtype alpha-Syn also are implicated in the pathogenesis of sporadic PD, the most common movement disorder. Insights into mechanisms underlying alpha-Syn mediated neurodegeneration link alpha-Syn oligomerization and fibrillization to the onset and progression of PD. Thus, inhibiting alpha-Syn oligomer or fibril formation is a compelling target for discovering disease modifying therapies for PD, DLB, and related synucleinopathies. Although amyloid dyes recognize alpha-Syn fibrils, efficient detection of soluble oligomers remains a challenge. Here, we report a novel fluorescence polarization (FP) technique for examining alpha-Syn assembly by monitoring changes in its relative molecular mass during progression of normal alpha-Syn from highly soluble monomers to higher order multimers and thence insoluble amyloid fibrils. We report that FP is more sensitive than conventional amyloid dye methods for the quantification of mature fibrils, and that FP is capable of detecting oligomeric alpha-Syn, allowing for rapid automated screening of potential inhibitors of alpha-Syn oligomerization and fibrillization. Furthermore, FP can be combined with an amyloid dye in a single assay that simultaneously provides two independent biophysical readouts for monitoring alpha-Syn fibrillization. Thus, this FP method holds potential to accelerate discovery of disease modifying therapies for LB PD, DLB, and related neurodegenerative synucleinopathies.

Vitamin A potently destabilizes preformed alpha-synuclein fibrils in vitro: implications for Lewy body diseases.

Alpha-synuclein (alphaS) is the major component of the filamentous inclusions that constitute defining characteristics of Lewy body diseases (LBD) and multiple system atrophy (MSA). Clinically, antioxidant vitamins, such as vitamin E and the vitamin-like substance coenzyme Q10, have been used in the treatment of LBD with some efficacy. Using fluorescence spectroscopy with thioflavin S, electron microscopy and atomic force microscopy, here we examined the effects of ten antioxidant vitamins and vitamin-like substances, vitamin A (retinol, retinal and retinoic acid), beta-carotene, vitamins B2, B6, C, E, coenzyme Q10 and alpha-lipoic acid, on the formation of alphaS fibrils (falphaS) and on preformed falphaS. Among them, vitamin A, beta-carotene and coenzyme Q10 dose-dependently inhibited the formation of falphaS. Moreover, they also dose-dependently destabilized preformed falphaS. With such potent anti-fibrillogenic as well as fibril-destabilizing activities, these compounds could be useful in the treatment and prevention of LBD and MSA.

Tau-dependent microtubule disassembly initiated by prefibrillar beta-amyloid.

Alzheimer's Disease (AD) is defined histopathologically by extracellular beta-amyloid (Abeta) fibrils plus intraneuronal tau filaments. Studies of transgenic mice and cultured cells indicate that AD is caused by a pathological cascade in which Abeta lies upstream of tau, but the steps that connect Abeta to tau have remained undefined. We demonstrate that tau confers acute hypersensitivity of microtubules to prefibrillar, extracellular Abeta in nonneuronal cells that express transfected tau and in cultured neurons that express endogenous tau. Prefibrillar Abeta42 was active at submicromolar concentrations, several-fold below those required for equivalent effects of prefibrillar Abeta40, and microtubules were insensitive to fibrillar Abeta. The active region of tau was localized to an N-terminal domain that does not bind microtubules and is not part of the region of tau that assembles into filaments. These results suggest that a seminal cell biological event in AD pathogenesis is acute, tau-dependent loss of microtubule integrity caused by exposure of neurons to readily diffusible Abeta.

Inhibition of the formation of amyloid beta-protein fibrils using biocompatible nanogels as artificial chaperones.

The formation of fibrils by amyloid beta-protein (Abeta) is considered as a key step in the pathology of Alzheimer's disease (AD). Inhibiting the aggregation of Abeta is a promising approach for AD therapy. In this study, we used biocompatible nanogels composed of a polysaccharide pullulan backbone with hydrophobic cholesterol moieties (cholesterol-bearing pullulan, CHP) as artificial chaperones to inhibit the formation of Abeta-(1-42) fibrils with marked amyloidgenic activity and cytotoxicity. The CHP-nanogels incorporated up to 6-8 Abeta-(1-42) molecules per particle and induced a change in the conformation of Abeta from a random coil to alpha-helix- or beta-sheet-rich structure. This structure was stable even after a 24-h incubation at 37 degrees C and the aggregation of Abeta-(1-42) was suppressed. Furthermore, the dissociation of the nanogels caused by the addition of methyl-beta-cyclodextrin released monomeric Abeta molecules. Nanogels composed of amino-group-modified CHP (CHPNH(2)) with positive charges under physiological conditions had a greater inhibitory effect than CHP-nanogels, suggesting the importance of electrostatic interactions between CHPNH(2) and Abeta for inhibiting the formation of fibrils. In addition, CHPNH(2) nanogels protected PC12 cells from Abeta toxicity.

Bacopa monniera extract reduces amyloid levels in PSAPP mice.

PSAPP mice expressing the "Swedish" amyloid precursor protein and M146L presenilin-1 mutations are a well-characterized model for spontaneous amyloid plaque formation. Bacopa monniera has a long history of use in India as an anti-aging and memory-enhancing ethnobotanical therapy. To evaluate the effect of Bacopa monniera extract (BME) on amyloid (Abeta) pathology in PSAPP mice, two doses of BME (40 or 160 mg/kg/day) were administered starting at 2 months of age for either 2 or 8 months. Our present data suggests that BME lowers Abeta 1-40 and 1-42 levels in cortex by as much as 60%, and reverses Y-maze performance and open field hyperlocomotion behavioral changes present in PSAPP mice. The areas encompassed by Congo Red-positive fibrillar amyloid deposits, however, were not altered by BME treatment. The data suggest that BME has potential application in Alzheimer's disease therapeutics.

TMAO promotes fibrillization and microtubule assembly activity in the C-terminal repeat region of tau.

Alzheimer's disease most closely correlates with the appearance of the neurofibrillary tangles (NFTs), intracellular fibrous aggregates of the microtubule-associated protein, tau. Under native conditions, tau is an unstructured protein, and its physical characterization has revealed no clues about the three-dimensional structural determinants essential for aggregation or microtubule binding. We have found that the natural osmolyte trimethylamine N-oxide (TMAO) induces secondary structure in a C-terminal fragment of tau (tau(187)) and greatly promotes both self-aggregation and microtubule (MT) assembly activity. These processes could be distinguished, however, by a single-amino acid substitution (Tyr(310) --> Ala), which severely inhibited aggregation but had no effect on MT assembly activity. The inability of this mutant to aggregate could be completely reversed by TMAO. We propose a model in which TMAO induces partial order in tau(187), resulting in conformers that may correspond to on-pathway intermediates of either aggregation or tau-dependent MT assembly or both. These studies set the stage for future high-resolution structural characterization of these intermediates and the basis by which Tyr(310) may direct pathologic versus normal tau function.

Resveratrol prolongs lifespan and retards the onset of age-related markers in a short-lived vertebrate.

Resveratrol, a natural phytoalexin found in grapes and red wine, increases longevity in the short-lived invertebrates Caenorhabditis elegans and Drosophila and exerts a variety of biological effects in vertebrates, including protection from ischemia and neurotoxicity. Its effects on vertebrate lifespan were not yet known. The relatively long lifespan of mice, which live at least 2.5 years, is a hurdle for life-long pharmacological trials. Here, the authors used the short-lived seasonal fish Nothobranchius furzeri with a maximum recorded lifespan of 13 weeks in captivity. Short lifespan in this species is not the result of spontaneous or targeted genetic mutations, but a natural trait correlated with the necessity to breed in an ephemeral habitat and tied with accelerated development and expression of ageing biomarkers at a cellular level. Resveratrol was added to the food starting in early adulthood and caused a dose-dependent increase of median and maximum lifespan. In addition, resveratrol delays the age-dependent decay of locomotor activity and cognitive performances and reduces the expression of neurofibrillary degeneration in the brain. These results demonstrate that food supplementation with resveratrol prolongs lifespan and retards the expression of age-dependent traits in a short-lived vertebrate.

Inhibitory effect of minocycline on amyloid beta fibril formation and human microglial activation.

Minocycline, a derivative of the antibiotic tetracycline, displays neuroprotective properties in various models of neurodegenerative diseases and is now used in clinical trials, because of its relative safety and tolerability. Minocycline passes the blood-brain barrier and is presumed to inhibit microglial activation. In Alzheimer's disease brain, a number of proteins, including serum amyloid P component (SAP) and complement factors such as C1q, accumulate in amyloid beta (Abeta) plaques. In a previous study, SAP and C1q were found to be required for clustering of activated microglia in Abeta plaques. Furthermore, SAP and C1q enhanced Abeta fibril formation and Abeta mediated cytokine release by human microglia in vitro. In the present study, we report that tetracycline and minocycline dose-dependently reduce TNF-alpha and IL-6 release by adult human microglia upon stimulation with a combination of Abeta, SAP, and C1q. In addition, minocycline and to a lesser extent tetracycline inhibit fibril formation of Abeta as determined in a thioflavin-S-based fluorescence test. This inhibitory effect was observed with Abeta alone as well as with Abeta in combination with SAP and C1q. Our data suggest that minocycline and tetracycline at tolerable doses can inhibit human microglial activation. This activity in part is exerted by inhibition of (SAP and C1q enhanced) Abeta fibril formation.