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.     

Identification of Novel GSK-3β Hits Using Competitive Biophysical Assays

Glycogen synthase kinase 3 beta (GSK-3β) is an evolutionarily conserved serine-threonine kinase dysregulated in numerous pathologies, such as Alzheimer’s disease and cancer. Even though GSK-3β is a validated pharmacological target most of its inhibitors have two main limitations: the lack of selectivity due to the high homology that characterizes the ATP binding site of most kinases, and the toxicity that emerges from GSK-3β complete inhibition which translates into the impairment of the plethora of pathways GSK-3β is involved in. Starting from a 1D ¹⁹F NMR fragment screening, we set up several biophysical assays for the identification of GSK-3β inhibitors capable of binding protein hotspots other than the ATP binding pocket or to the ATP binding pocket, but with an affinity able of competing with a reference binder. A phosphorylation activity assay on a panel of several kinases provided selectivity data that were further rationalized and corroborated by structural information on GSK-3β in complex with the hit compounds. In this study, we identified promising fragments, inhibitors of GSK-3β, while proposing an alternative screening workflow that allows facing the flaws that characterize the most common GSK-3β inhibitors through the identification of selective inhibitors and/or inhibitors able to modulate GSK-3β activity without leading to its complete inhibition.     

A Multichannel Fluorescent Tongue for Amyloid-β Aggregates Detection

Attention has been paid to the early diagnosis of Alzheimer’s disease, due to the maximum benefit acquired from the early-stage intervention and treatment. However, the sensing techniques primarily depended upon for neuroimaging and immunological assays for the detection of AD biomarkers are expensive, time-consuming and instrument dependent. Here, we developed a multichannel fluorescent tongue consisting of four fluorescent dyes and GO through electrostatic and π–π interaction. The array distinguished multiple aggregation states of 1 µM Aβ40/Aβ42 with 100% prediction accuracy via 10-channel signal outputs, illustrating the rationality of the array design. Screening vital sensor elements for the simplified sensor array and the optimization of sensing system was achieved by machine learning algorithms. Moreover, our sensing tongue was able to detect the aggregation states of Aβ40/Aβ42 in serum, demonstrating the great potential of multichannel array in diagnosing the Alzheimer’s diseases.     

Impact of Amyloid-β on Platelet Mitochondrial Function and Platelet–Mediated Amyloid Aggregation in Alzheimer’s Disease

Background: Alzheimer’s disease (AD) is characterized by an accumulation of amyloid β (Aβ) peptides in the brain and mitochondrial dysfunction. Platelet activation is enhanced in AD and platelets contribute to AD pathology by their ability to facilitate soluble Aβ to form Aβ aggregates. Thus, anti-platelet therapy reduces the formation of cerebral amyloid angiopathy in AD transgenic mice. Platelet mitochondrial dysfunction plays a regulatory role in thrombotic response, but its significance in AD is unknown and explored herein. Methods: The effects of Aβ-mediated mitochondrial dysfunction in platelets were investigated in vitro. Results: Aβ40 stimulation of human platelets led to elevated reactive oxygen species (ROS) and superoxide production, while reduced mitochondrial membrane potential and oxygen consumption rate. Enhanced mitochondrial dysfunction triggered platelet-mediated Aβ40 aggregate formation through GPVI-mediated ROS production, leading to enhanced integrin αII_bβ₃ activation during synergistic stimulation from ADP and Aβ40. Aβ40 aggregate formation of human and murine (APP23) platelets were comparable to controls and could be reduced by the antioxidant vitamin C. Conclusions: Mitochondrial dysfunction contributes to platelet-mediated Aβ aggregate formation and might be a promising target to limit platelet activation exaggerated pathological manifestations in AD.     

Interactions of Amyloid-β with Membrane Proteins

In developing and developed countries, an increasing elderly population is observed. This affects the growing percentage of people struggling with neurodegenerative diseases, including Alzheimer’s disease. Nevertheless, the pathomechanism of this disease is still unknown. This contributes to problems with early diagnosis of the disease as well as with treatment. One of the most popular hypotheses of Alzheimer’s disease is related to the pathological deposition of amyloid-β (Aβ) in the brain of ill people. In this paper, we discuss issues related to Aβ and its relationship in the development of Alzheimer’s disease. The structure of Aβ and its interaction with the cell membrane are discussed. Not only do the extracellular plaques affect nerve cells, but other forms of this peptide as well.     

Trans-Chalcone Plus Baicalein Synergistically Reduce Intracellular Amyloid Beta (Aβ42) and Protect from Aβ42 Induced Oxidative Damage in Yeast Models of Alzheimer’s Disease

Finding an effective therapeutic to prevent or cure AD has been difficult due to the complexity of the brain and limited experimental models. This study utilized unmodified and genetically modified Saccharomyces cerevisiae as model organisms to find potential natural bioactive compounds capable of reducing intracellular amyloid beta 42 (Aβ₄₂) and associated oxidative damage. Eleven natural bioactive compounds including mangiferin, quercetin, rutin, resveratrol, epigallocatechin gallate (EGCG), urolithin A, oleuropein, rosmarinic acid, salvianolic acid B, baicalein and trans-chalcone were screened for their ability to reduce intracellular green fluorescent protein tagged Aβ₄₂ (GFP-Aβ₄₂) levels. The two most effective compounds from the screens were combined in varying concentrations of each to study the combined capacity to reduce GFP-Aβ₄₂. The most effective combinations were examined for their effect on growth rate, turnover of native Aβ₄₂ and reactive oxygen species (ROS). The bioactive compounds except mangiferin and urolithin A significantly reduced intracellular GFP-Aβ₄₂ levels. Baicalein and trans-chalcone were the most effective compounds among those that were screened. The combination of baicalein and trans-chalcone synergistically reduced GFP-Aβ₄₂ levels. A combination of 15 μM trans-chalcone and 8 μM baicalein was found to be the most synergistic combination. The combination of the two compounds significantly reduced ROS and Aβ₄₂ levels in yeast cells expressing native Aβ₄₂ without affecting growth of the cells. These findings suggest that the combination of baicalein and trans-chalcone could be a promising multifactorial therapeutic strategy to cure or prevent AD. However, further studies are recommended to look for similar cytoprotective activity in humans and to find an optimal dosage.     

From Brain to Heart: Possible Role of Amyloid-β in Ischemic Heart Disease and Ischemia-Reperfusion Injury

Ischemic heart disease (IHD) is among the leading causes of death in developed countries. Its pathological origin is traced back to coronary atherosclerosis, a lipid-driven immuno-inflammatory disease of the arteries that leads to multifocal plaque development. The primary clinical manifestation of IHD is acute myocardial infarction (AMI),) whose prognosis is ameliorated with optimal timing of revascularization. Paradoxically, myocardium re-perfusion can be detrimental because of ischemia-reperfusion injury (IRI), an oxidative-driven process that damages other organs. Amyloid-β (Aβ) plays a physiological role in the central nervous system (CNS). Alterations in its synthesis, concentration and clearance have been connected to several pathologies, such as Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA). Aβ has been suggested to play a role in the pathogenesis of IHD and cerebral IRI. The purpose of this review is to summarize what is known about the pathological role of Aβ in the CNS; starting from this evidence, we will illustrate the role played by Aβ in the development of coronary atherosclerosis and its possible implications in the pathophysiology of IHD and myocardial IRI. Better elucidation of Aβ’s contribution to the molecular pathways underlying IHD and IRI could be of great help in developing new therapeutic strategies.     

Free Cholesterol Accelerates Aβ Self-Assembly on Membranes at Physiological Concentration

The effects of membranes on the early-stage aggregation of amyloid β (Aβ) have come to light as potential mechanisms by which neurotoxic species are formed in Alzheimer’s disease. We have shown that direct Aβ-membrane interactions dramatically enhance the Aβ aggregation, allowing for oligomer assembly at physiologically low concentrations of the monomer. Membrane composition is also a crucial factor in this process. Our results showed that apart from phospholipids composition, cholesterol in membranes significantly enhances the aggregation kinetics. It has been reported that free cholesterol is present in plaques. Here we report that free cholesterol, along with its presence inside the membrane, further accelerate the aggregation process by producing aggregates more rapidly and of significantly larger sizes. These aggregates, which are formed on the lipid bilayer, are able to dissociate from the surface and accumulate in the bulk solution; the presence of free cholesterol accelerates this dissociation as well. All-atom molecular dynamics simulations show that cholesterol binds Aβ monomers and significantly changes the conformational sampling of Aβ monomer; more than doubling the fraction of low-energy conformations compared to those in the absence of cholesterol, which can contribute to the aggregation process. The results indicate that Aβ-lipid interaction is an important factor in the disease prone amyloid assembly process.     

Enhancing the Amyloid-β Anti-Aggregation Properties of Curcumin via Arene-Ruthenium(II) Derivatization

Alzheimer’s disease (AD) is a fatal neurodegenerative disorder associated with severe dementia, progressive cognitive decline, and irreversible memory loss. Although its etiopathogenesis is still unclear, the aggregation of amyloid-β (Aβ) peptides into supramolecular structures and their accumulation in the central nervous system play a critical role in the onset and progression of the disease. On such a premise, the inhibition of the early stages of Aβ aggregation is a potential prevention strategy for the treatment of AD. Since several natural occurring compounds, as well as metal-based molecules, showed promising inhibitory activities toward Aβ aggregation, we herein characterized the interaction of an organoruthenium derivative of curcumin with Aβ(1–40) and Aβ(1–42) peptides, and we evaluated its ability to inhibit the oligomerization/fibrillogenesis processes by combining in silico and in vitro methods. In general, besides being less toxic to neuronal cells, the derivative preserved the amyloid binding ability of the parent compound in terms of equilibrium dissociation constants but (most notably) was more effective both in retarding the formation and limiting the size of amyloid aggregates by virtue of a higher hindering effect on the amyloid–amyloid elongation surface. Additionally, the complex protected neuronal cells from amyloid toxicity.     

Role of Retinal Amyloid-β in Neurodegenerative Diseases: Overlapping Mechanisms and Emerging Clinical Applications

Amyloid-β (Aβ) accumulations have been identified in the retina for neurodegeneration-associated disorders like Alzheimer’s disease (AD), glaucoma, and age-related macular degeneration (AMD). Elevated retinal Aβ levels were associated with progressive retinal neurodegeneration, elevated cerebral Aβ accumulation, and increased disease severity with a decline in cognition and vision. Retinal Aβ accumulation and its pathological effects were demonstrated to occur prior to irreversible neurodegeneration, which highlights its potential in early disease detection and intervention. Using the retina as a model of the brain, recent studies have focused on characterizing retinal Aβ to determine its applicability for population-based screening of AD, which warrants a further understanding of how Aβ manifests between these disorders. While current treatments directly targeting Aβ accumulations have had limited results, continued exploration of Aβ-associated pathological pathways may yield new therapeutic targets for preserving cognition and vision. Here, we provide a review on the role of retinal Aβ manifestations in these distinct neurodegeneration-associated disorders. We also discuss the recent applications of retinal Aβ for AD screening and current clinical trial outcomes for Aβ-associated treatment approaches. Lastly, we explore potential future therapeutic targets based on overlapping mechanisms of pathophysiology in AD, glaucoma, and AMD.     

New Evidence for P-gp-Mediated Export of Amyloid-β Peptides in Molecular,Blood-Brain Barrier and Neuronal Models

Defective clearance mechanisms lead to the accumulation of amyloid-beta (Aβ) peptides in the Alzheimer’s brain. Though predominantly generated in neurons, little is known about how these hydrophobic, aggregation-prone, and tightly membrane-associated peptides exit into the extracellular space where they deposit and propagate neurotoxicity. The ability for P-glycoprotein (P-gp), an ATP-binding cassette (ABC) transporter, to export Aβ across the blood-brain barrier (BBB) has previously been reported. However, controversies surrounding the P-gp–Aβ interaction persist. Here, molecular data affirm that both Aβ₄₀ and Aβ₄₂ peptide isoforms directly interact with and are substrates of P-gp. This was reinforced ex vivo by the inhibition of Aβ₄₂ transport in brain capillaries from P-gp-knockout mice. Moreover, we explored whether P-gp could exert the same role in neurons. Comparison between non-neuronal CHO-APP and human neuroblastoma SK-N-SH cells revealed that P-gp is expressed and active in both cell types. Inhibiting P-gp activity using verapamil and nicardipine impaired Aβ₄₀ and Aβ₄₂ secretion from both cell types, as determined by ELISA. Collectively, these findings implicate P-gp in Aβ export from neurons, as well as across the BBB endothelium, and suggest that restoring or enhancing P-gp function could be a viable therapeutic approach for removing excess Aβ out of the brain in Alzheimer’s disease.     

Amyloid-β Processing in Aged S100B Transgenic Mice Is Sex Dependent

(1) Background: Calcium-binding protein S100B is involved in neuroregeneration but has also been associated with neurodegeneration. These contrasting effects may result from concentration or duration of exposure. We investigated the effect of long-term increased S100B levels on amyloid-β processing in one-year-old transgenic (tg) mice with 12 copies of the murine S100B gene with specific consideration of sex and specific brain regions. (2) Methods: S100B and amyloid-β 42 (Aβ42) were quantified in serum, cerebrospinal fluid (CSF), adipose tissue, and different brain regions by ELISA in wild-type (wt) and S100Btg mice (each n = 7 per group). Thioflavin T (ThT) and Aβ immunostaining were performed for visualization of Aβ deposition. (3) Results: S100B in serum, CSF, and brain was significantly increased in S100Btg mice of both sexes. Aβ42 was significantly increased in the hippocampus of male S100Btg mice (p = 0.0075), and the frontal cortex of female S100Btg mice (p = 0.0262). ThT and Aβ immunostaining demonstrated Aβ deposition in different brain regions in S100Btg mice of both sexes and female wt. (4) Conclusion: Our data validate this experimental model for studying the role of S100B in neurodegeneration and indicate that Aβ processing is sex-dependent and brain region-specific, which deserves further investigation of signaling pathways and behavioral responses.     

Aβ-Targeting Bifunctional Chelators (BFCs) for Potential Therapeutic and PET Imaging Applications

Currently, more than 55 million people live with dementia worldwide, and there are nearly 10 million new cases every year. Alzheimer’s disease (AD) is the most common neurodegenerative disease resulting in personality changes, cognitive impairment, memory loss, and physical disability. Diagnosis of AD is often missed or delayed in clinical practice due to the fact that cognitive deterioration occurs already in the later stages of the disease. Thus, methods to improve early detection would provide opportunities for early treatment of disease. All FDA-approved PET imaging agents for Aβ plaques use short-lived radioisotopes such as ¹¹C (t_1/2 = 20.4 min) and ¹⁸F (t_1/2 = 109.8 min), which limit their widespread use. Thus, a novel metal-based imaging agent for visualization of Aβ plaques is of interest, due to the simplicity of its synthesis and the longer lifetimes of its constituent isotopes. We have previously summarized a metal-containing drug for positron emission tomography (PET), magnetic resonance imaging (MRI), and single-photon emission computed tomography (SPECT) imaging of Alzheimer’s disease. In this review, we have summarized a recent advance in design of Aβ-targeting bifunctional chelators for potential therapeutic and PET imaging applications, reported after our previous review.     

Ibuprofen Favors Binding of Amyloid-β Peptide to Its Depot,Serum Albumin

The deposition of amyloid-β peptide (Aβ) in the brain is a critical event in the progression of Alzheimer’s disease (AD). This Aβ deposition could be prevented by directed enhancement of Aβ binding to its natural depot, human serum albumin (HSA). Previously, we revealed that specific endogenous ligands of HSA improve its affinity to monomeric Aβ. We show here that an exogenous HSA ligand, ibuprofen (IBU), exerts the analogous effect. Plasmon resonance spectroscopy data evidence that a therapeutic IBU level increases HSA affinity to monomeric Aβ₄₀/Aβ₄₂ by a factor of 3–5. Using thioflavin T fluorescence assay and transmission electron microcopy, we show that IBU favors the suppression of Aβ₄₀ fibrillation by HSA. Molecular docking data indicate partial overlap between the IBU/Aβ₄₀-binding sites of HSA. The revealed enhancement of the HSA–Aβ interaction by IBU and the strengthened inhibition of Aβ fibrillation by HSA in the presence of IBU could contribute to the neuroprotective effects of the latter, previously observed in mouse and human studies of 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.     

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.     

Differential Clearance of Aβ Species from the Brain by Brain Lymphatic Endothelial Cells in Zebrafish

The failure of amyloid beta (Aβ) clearance is a major cause of Alzheimer’s disease, and the brain lymphatic systems play a crucial role in clearing toxic proteins. Recently, brain lymphatic endothelial cells (BLECs), a non-lumenized lymphatic cell in the vertebrate brain, was identified, but Aβ clearance via this novel cell is not fully understood. We established an in vivo zebrafish model using fluorescently labeled Aβ42 to investigate the role of BLECs in Aβ clearance. We discovered the efficient clearance of monomeric Aβ42 (mAβ42) compared to oligomeric Aβ42 (oAβ42), which was illustrated by the selective uptake of mAβ42 by BLECs and peripheral transport. The genetic depletion, pharmacological inhibition via the blocking of the mannose receptor, or the laser ablation of BLECs resulted in the defective clearance of mAβ42. The treatment with an Aβ disaggregating agent facilitated the internalization of oAβ42 into BLECs and improved the peripheral transport. Our findings reveal a new role of BLECs in the differential clearance of mAβ42 from the brain and provide a novel therapeutic strategy based on promoting Aβ clearance.     

Amyloid-Driven Tau Accumulation on Mitochondria Potentially Leads to Cognitive Deterioration in Alzheimer’s Disease

Despite the well-accepted role of the two main neuropathological markers (β-amyloid and tau) in the progression of Alzheimer’s disease, the interaction and specific contribution of each of them is not fully elucidated. To address this question, in the present study, an adeno-associated virus (AAV9) carrying the mutant P301L form of human tau, was injected into the dorsal hippocampi of APP/PS1 transgenic mice or wild type mice (WT). Three months after injections, memory tasks, biochemical and immunohistochemical analysis were performed. We found that the overexpression of hTauP301L accelerates memory deficits in APP/PS1 mice, but it did not affect memory function of WT mice. Likewise, biochemical assays showed that only in the case of APP/PS1-hTauP301L injected mice, an important accumulation of tau was observed in the insoluble urea fraction. Similarly, electron microscopy images revealed that numerous clusters of tau immunoparticles appear at the dendrites of APP/PS1 injected mice and not in WT animals, suggesting that the presence of amyloid is necessary to induce tau aggregation. Interestingly, these tau immunoparticles accumulate in dendritic mitochondria in the APP/PS1 mice, whereas most of mitochondria in WT injected mice remain free of tau immunoparticles. Taken together, it seems that amyloid induces tau aggregation and accumulation in the dendritic mitochondria and subsequently may alter synapse function, thus, contributing to accelerate cognitive decline in APP/PS1 mice.     

Near-Infrared Photothermally Enhanced Photo-Oxygenation for Inhibition of Amyloid-β Aggregation Based on RVG-Conjugated Porphyrinic Metal–Organic Framework and Indocyanine Green Nanoplatform

Amyloid aggregation is associated with many neurodegenerative diseases such as Alzheimer’s disease (AD). The current technologies using phototherapy for amyloid inhibition are usually photodynamic approaches based on evidence that reactive oxygen species can inhibit Aβ aggregation. Herein, we report a novel combinational photothermally assisted photo-oxygenation treatment based on a nano-platform of the brain-targeting peptide RVG conjugated with the 2D porphyrinic PCN−222 metal–organic framework and indocyanine green (PCN−222@ICG@RVG) with enhanced photo-inhibition in Alzheimer’s Aβ aggregation. A photothermally assisted photo-oxygenation treatment based on PCN@ICG could largely enhance the photo-inhibition effect on Aβ₄₂ aggregation and lead to much lower neurotoxicity upon near-infrared (NIR) irradiation at 808 nm compared with a single modality of photo-treatment in both cell-free and in vitro experiments. Generally, local photothermal heat increases the instability of Aβ aggregates and keeps Aβ in the status of monomers, which facilitates the photo-oxygenation process of generating oxidized Aβ monomers with low aggregation capability. In addition, combined with the brain-targeting peptide RVG, the PCN−222@ICG@RVG nanoprobe shows high permeability of the human blood–brain barrier (BBB) on a human brain-on-a-chip platform. The ex vivo study also demonstrates that NIR-activated PCN−222@ICG@RVG could efficiently dissemble Aβ plaques. Our work suggests that the combination of photothermal treatment with photo-oxygenation can synergistically enhance the inhibition of Aβ aggregation, which may boost NIR-based combinational phototherapy of AD in the future.