Cross-seeding of alpha-synuclein aggregation by amyloid fibrils of food proteins.

The aggregation of the protein α-synuclein (aSyn) into amyloid fibrils in the human brain is associated with the development of several neurodegenerative diseases, including Parkinson's disease. The previously observed prion-like spreading of aSyn aggregation throughout the brain and the finding that heterologous cross-seeding of amyloid aggregation occurs in vitro for some proteins suggest that exposure to amyloids in general may pose a risk for disease development. To elucidate which protein fibril characteristics determine if and how heterologous amyloid seeding can occur, we investigated the potential of amyloid fibrils formed from proteins found in food, hen egg white lysozyme, and bovine milk ß-lactoglobulin to cross-seed aSyn aggregation in the test tube. We observed that amyloid fibrils from lysozyme, but not ß-lactoglobulin, potently cross-seeded the aggregation of aSyn as indicated by a significantly shorter lag phase of aSyn aggregation in the presence of lysozyme fibrils. The cross-seeding effect of lysozyme was found to be primarily driven by a surface-mediated nucleation mechanism. The differential seeding effect of lysozyme and ß-lactoglobulin on aSyn aggregation could be explained on the basis of binding affinity, binding site, and electrostatic interactions. Our results indicate that heterologous seeding of proteins may occur depending on the physicochemical characteristics of the seed protein fibril. Our findings suggest that heterologous seeding has the potential to determine the pathogenesis of neurodegenerative amyloid diseases.

Alzheimer's Disease: Treatment Strategies and Their Limitations.

Alzheimer's disease (AD) is the most frequent case of neurodegenerative disease and is becoming a major public health problem all over the world. Many therapeutic strategies have been explored for several decades; however, there is still no curative treatment, and the priority remains prevention. In this review, we present an update on the clinical and physiological phase of the AD spectrum, modifiable and non-modifiable risk factors for AD treatment with a focus on prevention strategies, then research models used in AD, followed by a discussion of treatment limitations. The prevention methods can significantly slow AD evolution and are currently the best strategy possible before the advanced stages of the disease. Indeed, current drug treatments have only symptomatic effects, and disease-modifying treatments are not yet available. Drug delivery to the central nervous system remains a complex process and represents a challenge for developing therapeutic and preventive strategies. Studies are underway to test new techniques to facilitate the bioavailability of molecules to the brain. After a deep study of the literature, we find the use of soft nanoparticles, in particular nanoliposomes and exosomes, as an innovative approach for preventive and therapeutic strategies in reducing the risk of AD and solving problems of brain bioavailability. Studies show the promising role of nanoliposomes and exosomes as smart drug delivery systems able to penetrate the blood-brain barrier and target brain tissues. Finally, the different drug administration techniques for neurological disorders are discussed. One of the promising therapeutic methods is the intranasal administration strategy which should be used for preclinical and clinical studies of neurodegenerative diseases.

Searching for improved mimetic peptides inhibitors preventing conformational transition of amyloid-ß42 monomer.

A series of novel mimetic peptides were designed, synthesised and biologically evaluated as inhibitors of Aß42 aggregation. One of the synthesised peptidic compounds, termed compound 7 modulated Aß42 aggregation as demonstrated by thioflavin T fluorescence, acting also as an inhibitor of the cytotoxicity exerted by Aß42 aggregates. The early stage interaction between compound 7 and the Aß42 monomer was investigated by replica exchange molecular dynamics (REMD) simulations and docking studies. Our theoretical results revealed that compound 7 can elongate the helical conformation state of an early stage Aß42 monomer and it helps preventing the formation of ß-sheet structures by interacting with key residues in the central hydrophobic cluster (CHC). This strategy where early "on-pathway" events are monitored by small molecules will help the development of new therapeutic strategies for Alzheimer's disease.

Characterization of insulin-degrading enzyme-mediated cleavage of Aß in distinct aggregation states.

To enhance our understanding of the potential therapeutic utility of insulin-degrading enzyme (IDE) in Alzheimer's disease (AD), we studied in vitro IDE-mediated degradation of different amyloid-beta (Aß) peptide aggregation states. Our findings show that IDE activity is driven by the dynamic equilibrium between Aß monomers and higher ordered aggregates. We identify Met(35)-Val(36) as a novel IDE cleavage site in the Aß sequence and show that Aß fragments resulting from IDE cleavage form non-toxic amorphous aggregates. These findings need to be taken into account in therapeutic strategies designed to increase Aß clearance in AD patients by modulating IDE activity.

The mechanism of γ-Secretase dysfunction in familial Alzheimer disease.

The mechanisms by which mutations in the presenilins (PSEN) or the amyloid precursor protein (APP) genes cause familial Alzheimer disease (FAD) are controversial. FAD mutations increase the release of amyloid ß (Aß)42 relative to Aß40 by an unknown, possibly gain-of-toxic-function, mechanism. However, many PSEN mutations paradoxically impair γ-secretase and 'loss-of-function' mechanisms have also been postulated. Here, we use kinetic studies to demonstrate that FAD mutations affect Aß generation via three different mechanisms, resulting in qualitative changes in the Aß profiles, which are not limited to Aß42. Loss of ɛ-cleavage function is not generally observed among FAD mutants. On the other hand, γ-secretase inhibitors used in the clinic appear to block the initial ɛ-cleavage step, but unexpectedly affect more selectively Notch than APP processing, while modulators act as activators of the carboxypeptidase-like (γ) activity. Overall, we provide a coherent explanation for the effect of different FAD mutations, demonstrating the importance of qualitative rather than quantitative changes in the Aß products, and suggest fundamental improvements for current drug development efforts.

Two distinct ß-sheet structures in Italian-mutant amyloid-beta fibrils: a potential link to different clinical phenotypes.

Most Alzheimer's disease (AD) cases are late-onset and characterized by the aggregation and deposition of the amyloid-beta (Aß) peptide in extracellular plaques in the brain. However, a few rare and hereditary Aß mutations, such as the Italian Glu22-to-Lys (E22K) mutation, guarantee the development of early-onset familial AD. This type of AD is associated with a younger age at disease onset, increased ß-amyloid accumulation, and Aß deposition in cerebral blood vessel walls, giving rise to cerebral amyloid angiopathy (CAA). It remains largely unknown how the Italian mutation results in the clinical phenotype that is characteristic of CAA. We therefore investigated how this single point mutation may affect the aggregation of Aß1-42 in vitro and structurally characterized the resulting fibrils using a biophysical approach. This paper reports that wild-type and Italian-mutant Aß both form fibrils characterized by the cross-ß architecture, but with distinct ß-sheet organizations, resulting in differences in thioflavin T fluorescence and solvent accessibility. E22K Aß1-42 oligomers and fibrils both display an antiparallel ß-sheet structure, in comparison with the parallel ß-sheet structure of wild-type fibrils, characteristic of most amyloid fibrils described in the literature. Moreover, we demonstrate structural plasticity for Italian-mutant Aß fibrils in a pH-dependent manner, in terms of their underlying ß-sheet arrangement. These findings are of interest in the ongoing debate that (1) antiparallel ß-sheet structure might represent a signature for toxicity, which could explain the higher toxicity reported for the Italian mutant, and that (2) fibril polymorphism might underlie differences in disease pathology and clinical manifestation.

Could ecosystem management provide a new framework for Alzheimer's disease?

Alzheimer's disease (AD) is a progressive neurodegenerative brain disorder that involves a plethora of molecular pathways. In the context of therapeutic treatment and biomarker profiling, the amyloid-beta (Aß) peptide constitutes an interesting research avenue that involves interactions within a complex mixture of Aß alloforms and other disease-modifying factors. Here, we explore the potential of an ecosystem paradigm as a novel way to consider AD and Aß dynamics in particular. We discuss the example that the complexity of the Aß network not only exhibits interesting parallels with the functioning of complex systems such as ecosystems but that this analogy can also provide novel insights into the neurobiological phenomena in AD and serve as a communication tool. We propose that combining network medicine with general ecosystem management principles could be a new and holistic approach to understand AD pathology and design novel therapies.

Neurotoxicity of Alzheimer's disease Aß peptides is induced by small changes in the Aß42 to Aß40 ratio.

The amyloid peptides Aß(40) and Aß(42) of Alzheimer's disease are thought to contribute differentially to the disease process. Although Aß(42) seems more pathogenic than Aß(40), the reason for this is not well understood. We show here that small alterations in the Aß(42):Aß(40) ratio dramatically affect the biophysical and biological properties of the Aß mixtures reflected in their aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function tested in vitro and in vivo. A minor increase in the Aß(42):Aß(40) ratio stabilizes toxic oligomeric species with intermediate conformations. The initial toxic impact of these Aß species is synaptic in nature, but this can spread into the cells leading to neuronal cell death. The fact that the relative ratio of Aß peptides is more crucial than the absolute amounts of peptides for the induction of neurotoxic conformations has important implications for anti-amyloid therapy. Our work also suggests the dynamic nature of the equilibrium between toxic and non-toxic intermediates.

Mechanistic insights in glycation-induced protein aggregation.

Protein glycation causes loss-of-function through a process that has been associated with several diabetic-related diseases. Additionally, glycation has been hypothesized as a promoter of protein aggregation, which could explain the observed link between hyperglycaemia and the development of several aggregating diseases. Despite its relevance in a range of diseases, the mechanism through which glycation induces aggregation remains unknown. Here we describe the molecular basis of how glycation is linked to aggregation by applying a variety of complementary techniques to study the nonenzymatic glycation of hen lysozyme with ribose (ribosylation) as the reducing carbohydrate. Ribosylation involves a chemical multistep conversion that induces chemical modifications on lysine side chains without altering the protein structure, but changing the protein charge and enlarging its hydrophobic surface. These features trigger lysozyme native-like aggregation by forming small oligomers that evolve into bigger insoluble particles. Moreover, lysozyme incubated with ribose reduces the viability of SH-SY5Y neuroblastoma cells. Our new insights contribute toward a better understanding of the link between glycation and aggregation.

Biopurification using non-growing microorganisms to improve plant protein ingredients.

Securing a sustainable global food supply for a growing population requires a shift toward a more plant-based diet. The application of plant-based proteins is therefore increasing, but unpleasant off-flavors complicate their use. Here, we screened 97 microorganisms for their potential to remove off-flavors in a process with limiting amounts of fermentable sugar. This allowed the production of a more neutral-tasting, purified food ingredient while limiting microbial growth and the production of typical fermentation end products. We demonstrate that various lactic acid bacteria (LAB) and yeasts remove "green" aldehydes and ketones. This conversion can be carried out in less than one hour in almond, pea, potato, and oat proteins. Heterofermentative LAB was best at aldehyde and ketone neutralization with minimum de novo formation of microbial volatiles such as ethylacetate (sweet, fruity) or alpha-diketones (butter- and cheese-like). While sensory properties were improved, changes in protein solubility, emulsification, foaming, and in vitro digestibility were limited.

α-Galactosidase aggregation is a determinant of pharmacological chaperone efficacy on Fabry disease mutants.

Fabry disease is a lysosomal storage disorder caused by loss of α-galactosidase function. More than 500 Fabry disease mutants have been identified, the majority of which are structurally destabilized. A therapeutic strategy under development for lysosomal storage diseases consists of using pharmacological chaperones to stabilize the structure of the mutant protein, thereby promoting lysosomal delivery over retrograde degradation. The substrate analog 1-deoxygalactonojirimycin (DGJ) has been shown to restore activity of mutant α-galactosidase and is currently in clinical trial for treatment of Fabry disease. However, only ∼65% of tested mutants respond to treatment in cultured patient fibroblasts, and the structural underpinnings of DGJ response remain poorly explained. Using computational modeling and cell culture experiments, we show that the DGJ response is negatively affected by protein aggregation of α-galactosidase mutants, revealing a qualitative difference between misfolding-associated and aggregation-associated loss of function. A scoring function combining predicted thermodynamic stability and intrinsic aggregation propensity of mutants captures well their aggregation behavior under overexpression in HeLa cells. Interestingly, the same classifier performs well on DGJ response data of patient-derived cultured lymphoblasts, showing that protein aggregation is an important determinant of chemical chaperone efficiency under endogenous expression levels as well. Our observations reinforce the idea that treatment of aggregation-associated loss of function observed for the more severe α-galactosidase mutants could be enhanced by combining pharmacological chaperone treatment with the suppression of mutant aggregation, e.g. via proteostatic regulator compounds that increase cellular chaperone expression.

Molecular plasticity regulates oligomerization and cytotoxicity of the multipeptide-length amyloid-ß peptide pool.

Current therapeutic approaches under development for Alzheimer disease, including γ-secretase modulating therapy, aim at increasing the production of Aß(1-38) and Aß(1-40) at the cost of longer Aß peptides. Here, we consider the aggregation of Aß(1-38) and Aß(1-43) in addition to Aß(1-40) and Aß(1-42), in particular their behavior in mixtures representing the complex in vivo Aß pool. We demonstrate that Aß(1-38) and Aß(1-43) aggregate similar to Aß(1-40) and Aß(1-42), respectively, but display a variation in the kinetics of assembly and toxicity due to differences in short timescale conformational plasticity. In biologically relevant mixtures of Aß, Aß(1-38) and Aß(1-43) significantly affect the behaviors of Aß(1-40) and Aß(1-42). The short timescale conformational flexibility of Aß(1-38) is suggested to be responsible for enhancing toxicity of Aß(1-40) while exerting a cyto-protective effect on Aß(1-42). Our results indicate that the complex in vivo Aß peptide array and variations thereof is critical in Alzheimer disease, which can influence the selection of current and new therapeutic strategies.

Structural basis for increased toxicity of pathological aß42:aß40 ratios in Alzheimer disease.

The ß-amyloid peptide (Aß) is directly related to neurotoxicity in Alzheimer disease (AD). The two most abundant alloforms of the peptide co-exist under normal physiological conditions in the brain in an Aß(42):Aß(40) ratio of ∼1:9. This ratio is often shifted to a higher percentage of Aß(42) in brains of patients with familial AD and this has recently been shown to lead to increased synaptotoxicity. The molecular basis for this phenomenon is unclear. Although the aggregation characteristics of Aß(40) and Aß(42) individually are well established, little is known about the properties of mixtures. We have explored the biophysical and structural properties of physiologically relevant Aß(42):Aß(40) ratios by several techniques. We show that Aß(40) and Aß(42) directly interact as well as modify the behavior of the other. The structures of monomeric and fibrillar assemblies formed from Aß(40) and Aß(42) mixtures do not differ from those formed from either of these peptides alone. Instead, the co-assembly of Aß(40) and Aß(42) influences the aggregation kinetics by altering the pattern of oligomer formation as evidenced by a unique combination of solution nuclear magnetic resonance spectroscopy, high molecular weight mass spectrometry, and cross-seeding experiments. We relate these observations to the observed enhanced toxicity of relevant ratios of Aß(42):Aß(40) in synaptotoxicity assays and in AD patients.

Robust Tissue Fabrication for Long-Term Culture of iPSC-Derived Brain Organoids for Aging Research.

The currently available animal and cellular models do not fully recapitulate the complexity of changes that take place in the aging human brain. A recent development of procedures describing the generation of human cerebral organoids, derived from human induced pluripotent stem cells (iPSCs), has the potential to fundamentally transform the ability to model and understand the aging of the human brain and related pathogenic processes. Here, an optimized protocol for generating, maintaining, aging, and characterizing human iPSC-derived cerebral organoids is presented. This protocol can be implemented to generate brain organoids in a reproducible manner and serves as a step-by-step guide, incorporating the latest techniques that result in improved organoid maturation and aging in culture. Specific issues related to organoid maturation, necrosis, variability, and batch effects are being addressed. Taken together, these technological advances will allow the modeling of brain aging in organoids derived from a variety of young and aged human donors, as well as individuals afflicted with age-related brain disorders, allowing the identification of physiologic and pathogenic mechanisms of human brain aging.

The TNFR1 antagonist Atrosimab reduces neuronal loss, glial activation and memory deficits in an acute mouse model of neurodegeneration.

Tumor necrosis factor alpha (TNF-α) and its key role in modulating immune responses has been widely recognized as a therapeutic target for inflammatory and neurodegenerative diseases. Even though inhibition of TNF-α is beneficial for the treatment of certain inflammatory diseases, total neutralization of TNF-α largely failed in the treatment of neurodegenerative diseases. TNF-α exerts distinct functions depending on interaction with its two TNF receptors, whereby TNF receptor 1 (TNFR1) is associated with neuroinflammation and apoptosis and TNF receptor 2 (TNFR2) with neuroprotection and immune regulation. Here, we investigated the effect of administering the TNFR1-specific antagonist Atrosimab, as strategy to block TNFR1 signaling while maintaining TNFR2 signaling unaltered, in an acute mouse model for neurodegeneration. In this model, a NMDA-induced lesion that mimics various hallmarks of neurodegenerative diseases, such as memory loss and cell death, was created in the nucleus basalis magnocellularis and Atrosimab or control protein was administered centrally. We showed that Atrosimab attenuated cognitive impairments and reduced neuroinflammation and neuronal cell death. Our results demonstrate that Atrosimab is effective in ameliorating disease symptoms in an acute neurodegenerative mouse model. Altogether, our study indicates that Atrosimab may be a promising candidate for the development of a therapeutic strategy for the treatment of neurodegenerative diseases.

Use of standard U-bottom and V-bottom well plates to generate neuroepithelial embryoid bodies.

The use of organoids has become increasingly popular recently due to their self-organizing abilities, which facilitate developmental and disease modeling. Various methods have been described to create embryoid bodies (EBs) generated from embryonic or pluripotent stem cells but with varying levels of differentiation success and producing organoids of variable size. Commercial ultra-low attachment (ULA) V-bottom well plates are frequently used to generate EBs. These plates are relatively expensive and not as widely available as standard concave well plates. Here, we describe a cost-effective and low labor-intensive method that creates homogeneous EBs at high yield in standard V- and U-bottom well plates by applying an anti-adherence solution to reduce surface attachment, followed by centrifugation to enhance cellular aggregation. We also explore the effect of different seeding densities, in the range of 1 to 11 ×103 cells per well, for the fabrication of neuroepithelial EBs. Our results show that the use of V-bottom well plates briefly treated with anti-adherent solution (for 5 min at room temperature) consistently yields functional neural EBs in the range of seeding densities from 5 to 11×103 cells per well. A brief post-seeding centrifugation step further enhances EB establishment. EBs fabricated using centrifugation exhibited lower variability in their final size than their non-centrifuged counterparts, and centrifugation also improved EB yield. The span of conditions for reliable EB production is narrower in U-bottom wells than in V-bottom wells (i.e., seeding densities between 7×103 and 11×103 and using a centrifugation step). We show that EBs generated by the protocols introduced here successfully developed into neural organoids and expressed the relevant markers associated with their lineages. We anticipate that the cost-effective and easily implemented protocols presented here will greatly facilitate the generation of EBs, thereby further democratizing the worldwide ability to conduct organoid-based research.

A Review of Oxidative Stress Products and Related Genes in Early Alzheimer's Disease.

Oxidative stress is associated with the progression of Alzheimer's disease (AD). Reactive oxygen species can modify lipids, DNA, RNA, and proteins in the brain. The products of their peroxidation and oxidation are readily detectable at incipient stages of disease. Based on these oxidation products, various biomarker-based strategies have been developed to identify oxidative stress levels in AD. Known oxidative stress-related biomarkers include lipid peroxidation products F2-isoprostanes, as well as malondialdehyde and 4-hydroxynonenal which both conjugate to specific amino acids to modify proteins, and DNA or RNA oxidation products 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-hydroxyguanosine (8-OHG), respectively. The inducible enzyme heme oxygenase type 1 (HO-1) is found to be upregulated in response to oxidative stress-related events in the AD brain. While these global biomarkers for oxidative stress are associated with early-stage AD, they generally poorly differentiate from other neurodegenerative disorders that also coincide with oxidative stress. Redox proteomics approaches provided specificity of oxidative stress-associated biomarkers to AD pathology by the identification of oxidatively damaged pathology-specific proteins. In this review, we discuss the potential combined diagnostic value of these reported biomarkers in the context of AD and discuss eight oxidative stress-related mRNA biomarkers in AD that we newly identified using a transcriptomics approach. We review these genes in the context of their reported involvement in oxidative stress regulation and specificity for AD. Further research is warranted to establish the protein levels and their functionalities as well as the molecular mechanisms by which these potential biomarkers are involved in regulation of oxidative stress levels and their potential for determination of oxidative stress and disease status of AD patients.

Engineering Organoids for in vitro Modeling of Phenylketonuria.

Phenylketonuria is a recessive genetic disorder of amino-acid metabolism, where impaired phenylalanine hydroxylase function leads to the accumulation of neurotoxic phenylalanine levels in the brain. Severe cognitive and neuronal impairment are observed in untreated/late-diagnosed patients, and even early treated ones are not safe from life-long sequelae. Despite the wealth of knowledge acquired from available disease models, the chronic effect of Phenylketonuria in the brain is still poorly understood and the consequences to the aging brain remain an open question. Thus, there is the need for better predictive models, able to recapitulate specific mechanisms of this disease. Human induced pluripotent stem cells (hiPSCs), with their ability to differentiate and self-organize in multiple tissues, might provide a new exciting in vitro platform to model specific PKU-derived neuronal impairment. In this review, we gather what is known about the impact of phenylalanine in the brain of patients and highlight where hiPSC-derived organoids could contribute to the understanding of this disease.

Plant isoquinoline alkaloids as potential neurodrugs: A comparative study of the effects of benzo[c]phenanthridine and berberine-based compounds on ß-amyloid aggregation.

Herein we present a comparative study of the effects of isoquinoline alkaloids belonging to benzo[c]phenanthridine and berberine families on ß-amyloid aggregation. Results obtained using a Thioflavine T (ThT) fluorescence assay and circular dichroism (CD) spectroscopy suggested that the benzo[c]phenanthridine nucleus, present in both sanguinarine and chelerythrine molecules, was directly involved in an inhibitory effect of Aß1-42 aggregation. Conversely, coralyne, that contains the isomeric berberine nucleus, significantly increased propensity for Aß1-42 to aggregate. Surface Plasmon Resonance (SPR) experiments provided quantitative estimation of these interactions: coralyne bound to Aß1-42 with an affinity (KD = 11.6 µM) higher than benzo[c]phenanthridines. Molecular docking studies confirmed that all three compounds are able to recognize Aß1-42 in different aggregation forms suggesting their effective capacity to modulate the Aß1-42 self-recognition mechanism. Molecular dynamics simulations indicated that coralyne increased the ß-content of Aß1-42, in early stages of aggregation, consistent with fluorescence-based promotion of the Aß1-42 self-recognition mechanism by this alkaloid. At the same time, sanguinarine induced Aß1-42 helical conformation corroborating its ability to delay aggregation as experimentally proved in vitro. The investigated compounds were shown to interfere with aggregation of Aß1-42 demonstrating their potential as starting leads for the development of therapeutic strategies in neurodegenerative diseases.

Milk Processing Affects Structure, Bioavailability and Immunogenicity of ß-lactoglobulin.

Bovine milk is subjected to various processing steps to warrant constant quality and consumer safety. One of these steps is pasteurization, which involves the exposure of liquid milk to a high temperature for a limited amount of time. While such heating effectively ameliorates consumer safety concerns mediated by pathogenic bacteria, these conditions also have an impact on one of the main nutritional whey constituents of milk, the protein ß-lactoglobulin. As a function of heating, ß-lactoglobulin was shown to become increasingly prone to denaturation, aggregation, and lactose conjugation. This review discusses the implications of such heat-induced modifications on digestion and adsorption in the gastro-intestinal tract, and the responses these conformations elicit from the gastro-intestinal immune system.