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.

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.

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.

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.

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.

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.

Molecular Mechanisms and Genetics of Oxidative Stress in Alzheimer's Disease.

Alzheimer's disease is the most common neurodegenerative disorder that can cause dementia in elderly over 60 years of age. One of the disease hallmarks is oxidative stress which interconnects with other processes such as amyloid-ß deposition, tau hyperphosphorylation, and tangle formation. This review discusses current thoughts on molecular mechanisms that may relate oxidative stress to Alzheimer's disease and identifies genetic factors observed from in vitro, in vivo, and clinical studies that may be associated with Alzheimer's disease-related oxidative stress.

Apolipoprotein E associated with reconstituted high-density lipoprotein-like particles is protected from aggregation.

Apolipoprotein E (APOE) genotype determines Alzheimer's disease (AD) susceptibility, with the APOE ε4 allele being an established risk factor for late-onset AD. The ApoE lipidation status has been reported to impact amyloid-beta (Aß) peptide metabolism. The details of how lipidation affects ApoE behavior remain to be elucidated. In this study, we prepared lipid-free and lipid-bound ApoE particles, mimicking the high-density lipoprotein particles found in vivo, for all three isoforms (ApoE2, ApoE3, and ApoE4) and biophysically characterized them. We find that lipid-free ApoE in solution has the tendency to aggregate in vitro in an isoform-dependent manner under near-physiological conditions and that aggregation is impeded by lipidation of ApoE.

Benzodifurans for biomedical applications: BZ4, a selective anti-proliferative and anti-amyloid lead compound.

Aim: Our goal is to evaluate benzodifuran-based scaffolds for biomedical applications. Methodology: We here explored the anticancer and anti-amyloid activities of a novel compound (BZ4) in comparison with other known benzodifuran analogs, previously studied in our group, and we have explored its ability to interact with different DNA model systems. Results: BZ4 shows antiproliferative activity on different cancer cells; does not affect noncancerous control cells and alters the aggregation properties of ß-amyloid, as ascertained by circular dichroism, fluorescence spectroscopy and scanning electron microscopy analysis. An overall, qualitative picture on the mechanistic aspects related to the biological activities is discussed in light of the dynamic light scattering, UV, circular dichroism and fluorescence data, as well as of the metal ion-binding properties of BZ4.

Exposure of a cryptic Hsp70 binding site determines the cytotoxicity of the ALS-associated SOD1-mutant A4V.

The accumulation of toxic protein aggregates is thought to play a key role in a range of degenerative pathologies, but it remains unclear why aggregation of polypeptides into non-native assemblies is toxic and why cellular clearance pathways offer ineffective protection. We here study the A4V mutant of SOD1, which forms toxic aggregates in motor neurons of patients with familial amyotrophic lateral sclerosis (ALS). A comparison of the location of aggregation prone regions (APRs) and Hsp70 binding sites in the denatured state of SOD1 reveals that ALS-associated mutations promote exposure of the APRs more than the strongest Hsc/Hsp70 binding site that we could detect. Mutations designed to increase the exposure of this Hsp70 interaction site in the denatured state promote aggregation but also display an increased interaction with Hsp70 chaperones. Depending on the cell type, in vitro this resulted in cellular inclusion body formation or increased clearance, accompanied with a suppression of cytotoxicity. The latter was also observed in a zebrafish model in vivo. Our results suggest that the uncontrolled accumulation of toxic SOD1A4V aggregates results from insufficient detection by the cellular surveillance network.

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.

Structural and Aggregation Properties of Alpha-Synuclein Linked to Phospholipase A2 Action.

BACKGROUND: Alpha-synuclein is a protein involved in the pathogenesis of Parkinson's disease. In vitro observations have shown that specific brain-enriched polyunsaturated fatty acids, such as arachidonic acid, can give rise to a conformational change in alpha-synuclein and ultimately induce its fibrillation. Arachidonic acid is released by phospholipase A2 activity and clinical observations have shown a link between mutations in PLA2G6, the gene responsible for the production of phospholipase A2, and early-onset types of parkinsonism. It is unknown how phospholipase A2-driven release of arachidonic acid can affect the conformation of alphasynuclein. OBJECTIVE: The main objective of this study was to investigate if phospholipase A2-induced release of arachidonic acid can induce changes in conformation and aggregation state of alpha-synuclein. METHODS: Recombinant human alpha-synuclein was expressed and isolated and incubated in the presence of phosphatidylcholine and phosphatidylserine (PC/PS) containing liposomes. The release of free fatty acids from PC/PS liposomes by bee venom phospholipase A2 was measured with the fluorescent probe acrylodated intestinal fatty acid-binding protein (ADIFAB) and radioactive labelling by preparing liposomes in the presence of L- 3-phosphatidylcholine, 1-stearyl-2[1-14C] arachidonoyl. The effect of free fatty acid release on the conformation of alpha-synuclein was assayed by far-UV circular dichroism and resistance against V8 protease-induced limited proteolysis. Aggregation of alpha-synuclein upon exposure to phospholipase A2-induced action on PC/PS liposomes was measured using thioflavin T fluorescence, SDS-PAGE, gel filtration chromatography, and transmission electron microscopy. RAW264.7 cells were transiently transfected with human alpha-synuclein and release of arachidonic acid was quantified using radiolabeling and liquid scintillation counting. RESULTS: Phospholipase A2 is capable of releasing arachidonic acid from biomimetic phospholipid membranes. Exposure of alpha-synuclein to phospholipase A2-induced release of arachidonic acid from PC/PS liposomes induces a conformational transition of the protein and leads to partial resistance against proteolytic cleavage by V8 protease. Prolonged incubation of alpha-synuclein with arachidonic acid, derived from PC/PS liposomes by phospholipase A2 leads to aggregate formation. In line with this, transiently transfected RAW264.7 cells with alpha-synuclein showed arachidonic acid release and punctate alpha-synuclein staining upon phospholipase A2 activation. The ability of arachidonic acid to drive alpha-synuclein to aggregate was independent of its oxidation state. CONCLUSION: We present data that suggest a biological context for the previously reported clinical observation that linked mutations in PLA2G6, the gene responsible for the production of phospholipase A2, and early-onset types of parkinsonism. Release of arachidonic acid, independent of its oxidation state, through activation of phospholipase A2-driven hydrolysis of phospholipid membranes, leads to the structural transition and aggregation of alpha-synuclein.

The influence of N-terminal acetylation on micelle-induced conformational changes and aggregation of α-Synuclein.

The biological function of α-Synuclein has been related to binding to lipids and membranes but these interactions can also mediate α-Synuclein aggregation, which is associated to Parkinson's disease and other neuropathologies. In brain tissue α-Synuclein is constitutively N-acetylated, a modification that plays an important role in its conformational propensity, lipid and membrane binding, and aggregation propensity. We studied the interactions of the lipid-mimetic SDS with N-acetylated and non-acetylated α-Synuclein, as well as their early-onset Parkinson's disease variants A30P, E46K and A53T. At low SDS/protein ratios α-Synuclein forms oligomeric complexes with SDS micelles with relatively low α-helical structure. These micellar oligomers can efficiently nucleate aggregation of monomeric α-Synuclein, with successive formation of oligomers, protofibrils, curly fibrils and mature amyloid fibrils. N-acetylation reduces considerably the rate of aggregation of WT α-Synuclein. However, in presence of any of the early-onset Parkinson's disease mutations the protective effect of N-acetylation against micelle-induced aggregation becomes impaired. At higher SDS/protein ratios, N-acetylation favors another conformational transition, in which a second type of α-helix-rich, non-aggregating oligomers become stabilized. Once again, the Parkinson's disease mutations disconnect the influence of N-acetylation in promoting this transition. These results suggest a cooperative link between the N-terminus and the region of the mutations that may be important for α-Synuclein function.

Optimized Triton X-114 assisted lipopolysaccharide (LPS) removal method reveals the immunomodulatory effect of food proteins.

SCOPE: Investigations into the immunological response of proteins is often masked by lipopolysaccharide (LPS) contamination. We report an optimized Triton X-114 (TX-114) based LPS extraction method for ß-lactoglobulin (BLG) and soy protein extract suitable for cell-based immunological assays. METHODS AND RESULTS: Optimization of an existing TX-114 based phase LPS extraction method resulted in >99% reduction of LPS levels. However, remaining TX-114 was found to interfere with LPS and protein concentration assays and decreased viability of THP-1 macrophages and HEK-Blue 293 cells. Upon screening a range of TX-114 extraction procedures, TX-114-binding beads were found to most effectively lower TX-114 levels without affecting protein structural properties. LPS-purified proteins showed reduced capacity to activate TLR4 compared to non-treated proteins. LPS-purified BLG did not induce secretion of pro-inflammatory cytokines from THP-1 macrophages, as non-treated protein did, showing that LPS contamination masks the immunomodulatory effect of BLG. Both HEK293 cells expressing TLR4 and differentiated THP-1 macrophages were shown as a relevant model to screen the protein preparations for biological effects of LPS contamination. CONCLUSION: The reported TX-114 assisted LPS-removal from protein preparations followed by bead based removal of TX-114 allows evaluation of natively folded protein preparations for their immunological potential in cell-based studies.

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.

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.

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.