Cytotoxic Aβ Protofilaments Are Generated in the Process of Aβ Fibril Disaggregation

Significant research on Alzheimer’s disease (AD) has demonstrated that amyloid β (Aβ) oligomers are toxic molecules against neural cells. Thus, determining the generation mechanism of toxic Aβ oligomers is crucial for understanding AD pathogenesis. Aβ fibrils were reported to be disaggregated by treatment with small compounds, such as epigallocatechin gallate (EGCG) and dopamine (DA), and a loss of fibril shape and decrease in cytotoxicity were observed. However, the characteristics of intermediate products during the fibril disaggregation process are poorly understood. In this study, we found that cytotoxic Aβ aggregates are generated during a moderate disaggregation process of Aβ fibrils. A cytotoxicity assay revealed that Aβ fibrils incubated with a low concentration of EGCG and DA showed higher cytotoxicity than Aβ fibrils alone. Atomic force microscopy imaging and circular dichroism spectrometry showed that short and narrow protofilaments, which were highly stable in the β-sheet structure, were abundant in these moderately disaggregated samples. These results indicate that toxic Aβ protofilaments are generated during disaggregation from amyloid fibrils, suggesting that disaggregation of Aβ fibrils by small compounds may be one of the possible mechanisms for the generation of toxic Aβ aggregates in the brain.     

Combined Modeling Study of the Binding Characteristics of Natural Compounds,Derived from Psoralea Fruits,to β-Amyloid Peptide Monomer

A dysfunctional protein aggregation in the nervous system can lead to several neurodegenerative disorders that result in intracellular inclusions or extracellular aggregates. An early critical event within the pathogenesis of Alzheimer’s disease is the accumulation of amyloid beta peptide within the brain. Natural compounds isolated from Psoralea Fructus (PF) have significant anti-Alzheimer effects as strong inhibitors of Aβ42 aggregation. Computer simulations provide a powerful means of linking experimental findings to nanoscale molecular events. As part of this research four prenylated compounds, the active ingredients of Psoralea Fructus (PF), were studied as Aβ42 accumulation inhibitors using molecular simulations modeling. In order to resolve the binding modes of the ligands and identify the main interactions of Aβ42 residues, we performed a 100 ns molecular dynamics simulation and binding free energy calculations starting from the model of the compounds obtained from the docking study. This study was able to pinpoint the key amino acid residues in the Aβ42 active site and provide useful information that could benefit the development of new Aβ42 accumulation inhibitors.     

Oxidase Reactivity of CuII Bound to N-Truncated Aβ Peptides Promoted by Dopamine

The redox chemistry of copper(II) is strongly modulated by the coordination to amyloid-β peptides and by the stability of the resulting complexes. Amino-terminal copper and nickel binding motifs (ATCUN) identified in truncated Aβ sequences starting with Phe4 show very high affinity for copper(II) ions. Herein, we study the oxidase activity of [Cu–Aβ_4−x] and [Cu–Aβ_1−x] complexes toward dopamine and other catechols. The results show that the Cu^II–ATCUN site is not redox-inert; the reduction of the metal is induced by coordination of catechol to the metal and occurs through an inner sphere reaction. The generation of a ternary [Cu^II–Aβ–catechol] species determines the efficiency of the oxidation, although the reaction rate is ruled by reoxidation of the Cu^I complex. In addition to the N-terminal coordination site, the two vicinal histidines, His13 and His14, provide a second Cu-binding motif. Catechol oxidation studies together with structural insight from the mixed dinuclear complexes Ni/Cu–Aβ_4−x reveal that the His-tandem is able to bind Cu^II ions independently of the ATCUN site, but the N-terminal metal complexation reduces the conformational mobility of the peptide chain, preventing the binding and oxidative reactivity toward catechol of Cu^II bound to the secondary site.     

An Oligomannuronic Acid-Sialic Acid Conjugate Capable of Inhibiting Aβ42 Aggregation and Alleviating the Inflammatory Response of BV-2 Microglia

Oligomannuronic acid (MOS) from seaweed has antioxidant and anti-inflammatory activities. In this study, MOS was activated at the terminal to obtain three different graft complexes modified with sialic acid moiety (MOS-Sia). The results show that MOS-Sia addition can reduce the β-structure formation of Aβ42, and the binding effect of MOS-Sia3 is more obvious. MOS-Sia conjugates also have a better complexing effect with Ca²⁺ while reducing the formation of Aβ42 oligomers in solutions. MOS-Sia3 (25–50 μg/mL) can effectively inhibit the activation state of BV-2 cells stimulated by Aβ42, whereas a higher dose of MOS-Sia3 (>50 μg/mL) can inhibit the proliferation of BV-2 cells to a certain extent. A lower dose of MOS-Sia3 can also inhibit the expression of IL-1β, IL-6, TNF-α, and other proinflammatory factors in BV-2 cells induced by Aβ42 activation. In the future, the MOS-Sia3 conjugate can be used to treat Alzheimer’s disease.     

The Effects of Aβ1-42 Binding to the SARS-CoV-2 Spike Protein S1 Subunit and Angiotensin-Converting Enzyme 2

Increasing evidence suggests that elderly people with dementia are vulnerable to the development of severe coronavirus disease 2019 (COVID-19). In Alzheimer’s disease (AD), the major form of dementia, β-amyloid (Aβ) levels in the blood are increased; however, the impact of elevated Aβ levels on the progression of COVID-19 remains largely unknown. Here, our findings demonstrate that Aβ_1-42, but not Aβ_1-40, bound to various viral proteins with a preferentially high affinity for the spike protein S1 subunit (S1) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the viral receptor, angiotensin-converting enzyme 2 (ACE2). These bindings were mainly through the C-terminal residues of Aβ_1-42. Furthermore, Aβ_1-42 strengthened the binding of the S1 of SARS-CoV-2 to ACE2 and increased the viral entry and production of IL-6 in a SARS-CoV-2 pseudovirus infection model. Intriguingly, data from a surrogate mouse model with intravenous inoculation of Aβ_1-42 show that the clearance of Aβ_1-42 in the blood was dampened in the presence of the extracellular domain of the spike protein trimers of SARS-CoV-2, whose effects can be prevented by a novel anti-Aβ antibody. In conclusion, these findings suggest that the binding of Aβ_1-42 to the S1 of SARS-CoV-2 and ACE2 may have a negative impact on the course and severity of SARS-CoV-2 infection. Further investigations are warranted to elucidate the underlying mechanisms and examine whether reducing the level of Aβ_1-42 in the blood is beneficial to the fight against COVID-19 and AD.     

Exploring Aβ Proteotoxicity and Therapeutic Candidates Using Drosophila melanogaster

Alzheimer’s disease is a widespread and devastating neurological disorder associated with proteotoxic events caused by the misfolding and aggregation of the amyloid-β peptide. To find therapeutic strategies to combat this disease, Drosophila melanogaster has proved to be an excellent model organism that is able to uncover anti-proteotoxic candidates due to its outstanding genetic toolbox and resemblance to human disease genes. In this review, we highlight the use of Drosophila melanogaster to both study the proteotoxicity of the amyloid-β peptide and to screen for drug candidates. Expanding the knowledge of how the etiology of Alzheimer’s disease is related to proteotoxicity and how drugs can be used to block disease progression will hopefully shed further light on the field in the search for disease-modifying treatments.     

Amyloid β, Lipid Metabolism,Basal Cholinergic System,and Therapeutics in Alzheimer’s Disease

The presence of insoluble aggregates of amyloid β (Aβ) in the form of neuritic plaques (NPs) is one of the main features that define Alzheimer’s disease. Studies have suggested that the accumulation of these peptides in the brain significantly contributes to extensive neuronal loss. Furthermore, the content and distribution of cholesterol in the membrane have been shown to have an important effect on the production and subsequent accumulation of Aβ peptides in the plasma membrane, contributing to dysfunction and neuronal death. The monomeric forms of these membrane-bound peptides undergo several conformational changes, ranging from oligomeric forms to beta-sheet structures, each presenting different levels of toxicity. Aβ peptides can be internalized by particular receptors and trigger changes from Tau phosphorylation to alterations in cognitive function, through dysfunction of the cholinergic system. The goal of this review is to summarize the current knowledge on the role of lipids in Alzheimer’s disease and their relationship with the basal cholinergic system, as well as potential disease-modifying therapies.     

Diterpenoid Caesalmin C Delays Aβ-Induced Paralysis Symptoms via the DAF-16 Pathway in Caenorhabditis elegans

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease in the world. However, there is no effective drug to cure it. Caesalmin C is a cassane-type diterpenoid abundant in Caesalpinia bonduc (Linn.) Roxb. In this study, we investigated the effect of caesalmin C on Aβ-induced toxicity and possible mechanisms in the transgenic Caenorhabditis elegans AD model. Our results showed that caesalmin C significantly alleviated the Aβ-induced paralysis phenotype in transgenic CL4176 strain C. elegans. Caesalmin C dramatically reduced the content of Aβ monomers, oligomers, and deposited spots in AD C. elegans. In addition, mRNA levels of sod-3, gst-4, and rpt-3 were up-regulated, and mRNA levels of ace-1 were down-regulated in nematodes treated with caesalmin C. The results of the RNAi assay showed that the inhibitory effect of caesalmin C on the nematode paralysis phenotype required the DAF-16 signaling pathway, but not SKN-1 and HSF-1. Further evidence suggested that caesalmin C may also have the effect of inhibiting acetylcholinesterase (AchE) and upregulating proteasome activity. These findings suggest that caesalmin C delays the progression of AD in C. elegans via the DAF-16 signaling pathway and that it could be developed into a promising medication to treat AD.     

TNF-α and IL-1β Modulate Blood-Brain Barrier Permeability and Decrease Amyloid-β Peptide Efflux in a Human Blood-Brain Barrier Model

The blood-brain barrier (BBB) is a selective barrier and a functional gatekeeper for the central nervous system (CNS), essential for maintaining brain homeostasis. The BBB is composed of specialized brain endothelial cells (BECs) lining the brain capillaries. The tight junctions formed by BECs regulate paracellular transport, whereas transcellular transport is regulated by specialized transporters, pumps and receptors. Cytokine-induced neuroinflammation, such as the tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), appear to play a role in BBB dysfunction and contribute to the progression of Alzheimer’s disease (AD) by contributing to amyloid-β (Aβ) peptide accumulation. Here, we investigated whether TNF-α and IL-1β modulate the permeability of the BBB and alter Aβ peptide transport across BECs. We used a human BBB in vitro model based on the use of brain-like endothelial cells (BLECs) obtained from endothelial cells derived from CD34+ stem cells cocultivated with brain pericytes. We demonstrated that TNF-α and IL-1β differentially induced changes in BLECs’ permeability by inducing alterations in the organization of junctional complexes as well as in transcelluar trafficking. Further, TNF-α and IL-1β act directly on BLECs by decreasing LRP1 and BCRP protein expression as well as the specific efflux of Aβ peptide. These results provide mechanisms by which CNS inflammation might modulate BBB permeability and promote Aβ peptide accumulation. A future therapeutic intervention targeting vascular inflammation at the BBB may have the therapeutic potential to slow down the progression of AD.     

Chromeno[3,4-b]xanthones as First-in-Class AChE and Aβ Aggregation Dual-Inhibitors

Alzheimer’s disease (AD) is a complex multifactorial disorder, mainly characterized by the progressive loss of memory and cognitive, motor, and functional capacity. The absence of effective therapies available for AD alongside the consecutive failures in the central nervous system (CNS) drug development has been motivating the search for new disease-modifying therapeutic strategies for this disease. To address this issue, the multitarget directed ligands (MTDLs) are emerging as a therapeutic alternative to target the multiple AD-related factors. Following this concept, herein we describe the design, synthesis, and biological evaluation of a family of chromeno[3,4-b]xanthones as well as their (E)-2-[2-(propargyloxy)styryl]chromone precursors, as first-in-class acetylcholinesterase (AChE) and β-amyloid (Aβ) aggregation dual-inhibitors. Compounds 4b and 10 emerged as well-balanced dual-target inhibitors, with IC₅₀ values of 3.9 and 2.9 μM for AChE and inhibitory percentages of 70 and 66% for Aβ aggregation, respectively. The molecular docking showed that most of the compounds bound to AChE through hydrogen bonds with residues of the catalytic triad and π-stacking interactions between the main scaffold and the aromatic residues present in the binding pocket. The interesting well-balanced activities of these compounds makes them interesting templates for the development of new multitarget compounds for AD.     

Role of Intracellular Amyloid β as Pathway Modulator,Biomarker, and Therapy Target

The β- and γ-secretase-driven cleavage of the amyloid precursor protein (APP) gives rise to the amyloid β peptide, which is believed to be the main driver of neurodegeneration in Alzheimer’s disease (AD). As it is prominently detectable in extracellular plaques in post-mortem AD brain samples, research in recent decades focused on the pathological role of extracellular amyloid β aggregation, widely neglecting the potential meaning of very early generation of amyloid β inside the cell. In the last few years, the importance of intracellular amyloid β (iAβ) as a strong player in neurodegeneration has been indicated by a rising number of studies. In this review, iAβ is highlighted as a crucial APP cleavage fragment, able to manipulate intracellular pathways and foster neurodegeneration. We demonstrate its relevance as a pathological marker and shed light on initial studies aiming to modulate iAβ through pharmacological treatment, which has been shown to have beneficial effects on cognitive properties in animal models. Finally, we display the relevance of viral infections on iAβ generation and point out future directions urgently needed to manifest the potential relevance of iAβ in Alzheimer’s disease.     

Neurogenesis Is Increased in Human Neural Stem Cells by Aβ40 Peptide

Amyloid-β 40 peptides [Aβ1-40 (Aβ40)] are present within amyloid plaques in the brains of patients with Alzheimer’s disease (AD). Even though Aβ peptides are considered neurotoxic, they can mediate many biological processes, both in adult brains and throughout brain development. However, the physiological function of these Aβ peptides remains poorly understood, and the existing data are sometimes controversial. Here, we analyze and compare the effects of monomeric Aβ40 on the biology of differentiating human neural stem cells (human NSCs). For that purpose, we used a model of human NSCs called hNS1. Our data demonstrated that Aβ40 at high concentrations provokes apoptotic cellular death and the damage of DNA in human NSCs while also increasing the proliferation and favors neurogenesis by raising the percentage of proliferating neuronal precursors. These effects can be mediated, at least in part, by β-catenin. These results provide evidence of how Aβ modulate/regulate human NSC proliferation and differentiation, suggesting Aβ40 may be a pro-neurogenic factor. Our data could contribute to a better understanding of the molecular mechanisms involved in AD pathology and to the development of human NSC-based therapies for AD treatment, since these results could then be used in diagnosing the disease at early stages and be applied to the development of new treatment options.     

Disease-Modifying Effects of Non-Invasive Electroceuticals on β-Amyloid Plaques and Tau Tangles for Alzheimer’s Disease

Electroceuticals refer to various forms of electronic neurostimulators used for therapy. Interdisciplinary advances in medical engineering and science have led to the development of the electroceutical approach, which involves therapeutic agents that specifically target neural circuits, to realize precision therapy for Alzheimer’s disease (AD). To date, extensive studies have attempted to elucidate the disease-modifying effects of electroceuticals on areas in the brain of a patient with AD by the use of various physical stimuli, including electric, magnetic, and electromagnetic waves as well as ultrasound. Herein, we review non-invasive stimulatory systems and their effects on β-amyloid plaques and tau tangles, which are pathological molecular markers of AD. Therefore, this review will aid in better understanding the recent technological developments, applicable methods, and therapeutic effects of electronic stimulatory systems, including transcranial direct current stimulation, 40-Hz gamma oscillations, transcranial magnetic stimulation, electromagnetic field stimulation, infrared light stimulation and ionizing radiation therapy, and focused ultrasound for AD.     

Development of Alkaline Phosphatase-Fused Mouse Prion Protein and Its Application in Toxic Aβ Oligomer Detection

Amyloid β (Aβ) oligomers play a key role in the progression of Alzheimer’s disease (AD). Multiple forms of Aβ assemblies have been identified by in vitro and in vivo analyses; however, it is uncertain which oligomer is highly neurotoxic. Thus, understanding the pathogenesis of AD by detecting toxic Aβ oligomers is crucial. In this study, we report a fusion protein of cellular prion protein (PrPc) and alkaline phosphatase (ALP) from Escherichia coli as a sensing element for toxic Aβ oligomers. Since the N-terminus domain of PrPc (residue 23–111) derived from mice is known to bind to toxic Aβ oligomers in vitro, we genetically fused PrPc_23–111 to ALP. The developed fusion protein, PrP–ALP, retained both the binding ability of PrPc and enzymatic activity of ALP. We showed that PrP–ALP strongly bound to high molecular weight (HMW) oligomers but showed little or no affinity toward monomers. The observation that PrP–ALP neutralized the toxic effect of Aβ oligomers indicated an interaction between PrP–ALP and toxic HMW oligomers. Based on ALP activity, we succeeded in detecting Aβ oligomers. PrP–ALP may serve as a powerful tool for detecting toxic Aβ oligomers that may be related to AD progression.     

A Novel Cu(II)-Binding Peptide Identified by Phage Display Inhibits Cu2+-Mediated Aβ Aggregation

Copper (Cu) has been implicated in the progression of Alzheimer’s disease (AD), and aggregation of Cu and amyloid β peptide (Aβ) are considered key pathological features of AD. Metal chelators are considered to be potential therapeutic agents for AD because of their capacity to reduce metal ion-induced Aβ aggregation through the regulation of metal ion distribution. Here, we used phage display technology to screen, synthesize, and evaluate a novel Cu(II)-binding peptide that specifically blocked Cu-triggered Aβ aggregation. The Cu(II)-binding peptide (S-A-Q-I-A-P-H, PCu) identified from the phage display heptapeptide library was used to explore the mechanism of PCu inhibition of Cu²⁺-mediated Aβ aggregation and Aβ production. In vitro experiments revealed that PCu directly inhibited Cu²⁺-mediated Aβ aggregation and regulated copper levels to reduce biological toxicity. Furthermore, PCu reduced the production of Aβ by inhibiting Cu²⁺-induced BACE1 expression and improving Cu(II)-mediated cell oxidative damage. Cell culture experiments further demonstrated that PCu had relatively low toxicity. This Cu(II)-binding peptide that we have identified using phage display technology provides a potential therapeutic approach to prevent or treat AD.     

Combined Treatment with Curcumin and Ferulic Acid Suppressed the Aβ-Induced Neurotoxicity More than Curcumin and Ferulic Acid Alone

Alzheimer’s disease (AD) is a neurodegenerative disease that leads to progressive cognitive decline. Several effective natural components have been identified for the treatment of AD. However, it is difficult to obtain conclusive evidence on the safety and effectiveness of natural components, because a variety of factors are associated with the progression of AD pathology. We hypothesized that a therapeutic effect could be achieved by combining multiple ingredients with different efficacies. The purpose of this study was thus to evaluate a combination treatment of curcumin (Cur) and ferulic acid (FA) for amyloid-β (Aβ)-induced neuronal cytotoxicity. The effect of Cur or FA on Aβ aggregation using thioflavin T assay was confirmed to be inhibited in a concentration-dependent manner by Cur single or Cur + FA combination treatment. The effects of Cur + FA on the cytotoxicity of human neuroblastoma (SH-SY5Y) cells induced by Aβ exposure were an increase in cell viability, a decrease in ROS and mitochondrial ROS, and repair of membrane damage. Combination treatment showed an overall higher protective effect than treatment with Cur or FA alone. These results suggest that the combined action mechanisms of Cur and FA may be effective in preventing and suppressing the progression of AD.     

Dual Inhibitors of AChE and BACE-1 for Reducing Aβ in Alzheimer’s Disease: From In Silico to In Vivo

Alzheimer’s disease (AD) is a complex and widespread condition, still not fully understood and with no cure yet. Amyloid beta (Aβ) peptide is suspected to be a major cause of AD, and therefore, simultaneously blocking its formation and aggregation by inhibition of the enzymes BACE-1 (β-secretase) and AChE (acetylcholinesterase) by a single inhibitor may be an effective therapeutic approach, as compared to blocking one of these targets or by combining two drugs, one for each of these targets. We used our ISE algorithm to model each of the AChE peripheral site inhibitors and BACE-1 inhibitors, on the basis of published data, and constructed classification models for each. Subsequently, we screened large molecular databases with both models. Top scored molecules were docked into AChE and BACE-1 crystal structures, and 36 Molecules with the best weighted scores (based on ISE indexes and docking results) were sent for inhibition studies on the two enzymes. Two of them inhibited both AChE (IC₅₀ between 4–7 μM) and BACE-1 (IC₅₀ between 50–65 μM). Two additional molecules inhibited only AChE, and another two molecules inhibited only BACE-1. Preliminary testing of inhibition by F681-0222 (molecule 2) on APPswe/PS1dE9 transgenic mice shows a reduction in brain tissue of soluble Aβ42.     

Oligomeric and Fibrillar Species of Aβ42 Diversely Affect Human Neural Stem Cells

Amyloid-β 42 peptide (Aβ_1-42 (Aβ42)) is well-known for its involvement in the development of Alzheimer’s disease (AD). Aβ42 accumulates and aggregates in fibers that precipitate in the form of plaques in the brain causing toxicity; however, like other forms of Aβ peptide, the role of these peptides remains unclear. Here we analyze and compare the effects of oligomeric and fibrillary Aβ42 peptide on the biology (cell death, proliferative rate, and cell fate specification) of differentiating human neural stem cells (hNS1 cell line). By using the hNS1 cells we found that, at high concentrations, oligomeric and fibrillary Aβ42 peptides provoke apoptotic cellular death and damage of DNA in these cells, but Aβ42 fibrils have the strongest effect. The data also show that both oligomeric and fibrillar Aβ42 peptides decrease cellular proliferation but Aβ42 oligomers have the greatest effect. Finally, both, oligomers and fibrils favor gliogenesis and neurogenesis in hNS1 cells, although, in this case, the effect is more prominent in oligomers. All together the findings of this study may contribute to a better understanding of the molecular mechanisms involved in the pathology of AD and to the development of human neural stem cell-based therapies for AD treatment.     

Characterization of a Mouse Model of Alzheimer’s Disease Expressing Aβ4-42 and Human Mutant Tau

The relationship between the two most prominent neuropathological hallmarks of Alzheimer’s Disease (AD), extracellular amyloid-β (Aβ) deposits and intracellular accumulation of hyperphosphorylated tau in neurofibrillary tangles (NFT), remains at present not fully understood. A large body of evidence places Aβ upstream in the cascade of pathological events, triggering NFTs formation and the subsequent neuron loss. Extracellular Aβ deposits were indeed causative of an increased tau phosphorylation and accumulation in several transgenic models but the contribution of soluble Aβ peptides is still controversial. Among the different Aβ variants, the N-terminally truncated peptide Aβ_4–42 is among the most abundant. To understand whether soluble Aβ_4–42 peptides impact the onset or extent of tau pathology, we have crossed the homozygous Tg4–42 mouse model of AD, exclusively expressing Aβ_4–42 peptides, with the PS19 (P301S) tau transgenic model. Behavioral assessment showed that the resulting double-transgenic line presented a partial worsening of motor performance and spatial memory deficits in the aged group. While an increased loss of distal CA1 pyramidal neurons was detected in young mice, no significant alterations in hippocampal tau phosphorylation were observed in immunohistochemical analyses.