Synthesis of New Tyrosol-Based Phosphodiester Derivatives: Effect on Amyloid β Aggregation and Metal Chelation Ability

Alzheimer's disease (AD) is a multifactorial pathology that requires multifaceted agents able to address its peculiar nature. Increasing evidence has shown that aggregation of amyloid β (Aβ) and oxidative stress are strictly interconnected, and their modulation might have a positive and synergic effect in contrasting AD-related impairments. Herein, a new and efficient fragment-based approach towards tyrosol phosphodiester derivatives (TPDs) has been developed starting from suitable tyrosol building blocks and exploiting the well-established phosphoramidite chemistry. The antioxidant activity of new TPDs has been tested as well as their ability to inhibit Aβ protein aggregation. In addition, their metal chelating ability has been evaluated as a possible strategy to develop new natural-based entities for the prevention or therapy of AD. Interestingly, TPDs containing a catechol moiety have demonstrated highly promising activity in inhibiting the aggregation of Aβ₄₀ and a strong ability to chelate biometals such as Cu^II and Zn^II.     

Cerebrospinal Fluid Proteins as Regulators of Beta-amyloid Aggregation and Toxicity

Amyloid disorders, such as Alzheimer's, are almost invariably late-onset diseases. One defining diagnostic feature of Alzheimer's disease is the deposition of beta-amyloid as extracellular plaques, primarily in the hippocampus. This raises the question: are there natural protective agents that prevent beta-amyloid from depositing, and is it loss of this protection that leads to onset of disease? Proteins in cerebrospinal fluid (CSF) have been suggested to act as just such natural protective agents. Here, we describe some of the early evidence that led to this suggestion, and we discuss, in greater detail, two CSF proteins that have garnered the bulk of the attention.     

Fluorescent Probe DCVJ Shows High Sensitivity for Characterization of Amyloid β-Peptide Early in the Lag Phase

The aggregation of intrinsically disordered and misfolded proteins in the form of oligomers and fibrils plays a crucial role in a number of neurological and neurodegenerative diseases. Currently, most probes and biophysical techniques that detect and characterize fibrils at high resolution fail to show sensitivity and binding for oligomers. Here, we show that 9-(dicyano-vinyl)julolidine (DCVJ), a class of molecular rotor, binds amyloid beta (Aβ) early aggregates, and we report the kinetics as well as packing of the oligomer formation. The binding of DCVJ to Aβ40 increased its emission intensity with time at 510 nm and produced a second excimer peak at 575 nm. However, DCVJ did not bind to the prefibrillar aggregates of Aβ42, which indicated that the oligomers formed by Aβ40 and Aβ42 were not the same. The F4C F19W mutant of Aβ40, which did not form fibrils, also bound DCVJ, but the emission spectral profile varied from that of the wild-type (WT). Atomic force microscopy images of WT Aβ40, the F4C F19W mutant, and Aβ42 oligomers displayed differences in size and shape, confirming the difference in their DCVJ spectra. The effect of epigallocatechin-3-gallate (EGCG) on the reduction of Aβ42 fibrils was also observed with finer detail than with other techniques. The results of this study show that DCVJ detects early aggregates and provides valuable information regarding the oligomer kinetics, packing, and mechanism of formation.     

Compilation and Analysis of Enzymes,Engineered Antibodies,and Nanoparticles Designed to Interfere with Amyloid-β Aggregation

The abnormal accumulation and aggregation of amyloid β (Aβ) is one of the key factors of the synaptic impairment in Alzheimer's disease. Biomolecules, e.g., apolipoproteins, and membrane receptors, are implicated in the aggregation and toxicity of Aβ. Engineered molecules, such as enzymes, antibodies, and nanoparticles, are designed to interfere with these processes. We compile structural information on these molecules and their essential roles in the complex processes of aggregation, disaggregation, degradation, clearance, and inhibition of Aβ. The interactions between Aβ and its partners have no obvious emerging commonalities. One exception is the recognition of the N-terminal region of Aβ peptides by antibody heavy and light chains, which are facilitated by cooperative interaction not observed in other Aβ-peptide molecules. Overall, the emerging picture charts a diverse, to date unexplored, landscape and serves as the first-of-its-kind partner- and scenario-specific analysis.     

Perspectives on Inhibiting β‐Amyloid Aggregation through Structure‐Based Drug Design

Targeting β‐amyloid (Aβ) remains the most desired strategy in Alzheimer’s disease (AD) drug discovery research. Many peptides that specifically target Aβ aggregates are known, encompassing efforts from both industrial and academic research settings. However, in clinical terms, not much success has been gained with peptide research; in turn, small drug‐like molecules are already globally recognized as showing promise as an alternate approach. Aβ aggregation inhibitors are the most important part of the multifunctional drug design regimen for treating AD. Unfortunately, rational drug design approaches with small molecules are still in the initial stages. Herein we highlight, update, and elaborate on the structural anatomy of Aβ and known Aβ aggregation inhibitors in hopes of helping to optimize their use in structure‐based drug design approaches toward inhibitors with greater specificity. Furthermore, we present the first review of efforts to target a previously uncharacterized region of acetylcholinesterase: the N‐terminal 7–20 sub‐region, which was experimentally elucidated to participate in Aβ aggregation and deposition.     

Flexible N-Termini of Amyloid β-Protein Oligomers: A Link between Structure and Activity?

Revised amyloid cascade hypothesis of Alzheimer′s disease (AD) states that amyloid β-protein (Aβ) triggers the disease through formation of soluble low molecular weight (LMW) assemblies, called oligomers, which are challenging to characterize experimentally as well as computationally due to their heterogeneous and polymorphic nature, lack of ordered structure, and short lifetimes. Recent findings challenge the view of Aβ oligomers as exclusively toxic entities by revealing their dual, protective and disruptive nature in the context of immune response and AD, respectively. In this review, the understanding of Aβ oligomer formation and structure is discussed from the AD perspective. The structure-activity relationship (SAR) that implicates flexible, solvent exposed N-termini of Aβ oligomers, observed in computer simulations, in mediating their toxic as well as protective activity is proposed.     

Conformational Transitions of the Amyloid-β Peptide Upon Copper(II) Binding and pH Changes

Amyloid-β (Aβ) is a natively unfolded peptide found in all Alzheimer's disease patients as the major component of fibrillar plaques, which are recognized as an important pathological hallmark in Alzheimer's disease. The binding of copper to Aβ increases its neurotoxicity, as Cu²⁺ causes Aβ to become redox active and decreases the lag time associated with Aβ aggregation. In addition, the pH is a major factor that influences both the Aβ aggregation rates and Cu²⁺ binding. Hamiltonian replica exchange molecular dynamics (H-REMD) simulations enable atomistic insights into the effects of pH and Cu²⁺ complexation on the structure and dynamics of Aβ. To study the Aβ_1–42/Cu²⁺ complex, we have developed new force-field parameters for the divalent copper ion ligated by the two histidine residues, His6 and His13, as well as the amine and carbonyl groups of Asp1, in a distorted square-planar geometry. Our comparative simulations reveal that both Cu²⁺ binding and a low pH-mimicking acidosis, linked to inflammatory processes in vivo, accelerate the formation of β-strands in Aβ_1–42 and lead to the stabilization of salt bridges, previously shown to promote Aβ aggregation. The results suggest that Cu²⁺ binding and mild acidic conditions can shift the conformational equilibrium towards aggregation-prone conformers for the monomeric Aβ.     

Thiophene-Based Optical Ligands That Selectively Detect Aβ Pathology in Alzheimer's Disease

In several neurodegenerative diseases, the presence of aggregates of specific proteins in the brain is a significant pathological hallmark; thus, developing ligands able to bind to the aggregated proteins is essential for any effort related to imaging and therapeutics. Here we report the synthesis of thiophene-based ligands containing nitrogen heterocycles. The ligands selectively recognized amyloid-β (Aβ) aggregates in brain tissue from individuals diagnosed neuropathologically as having Alzheimer's disease (AD). The selectivity for Aβ was dependent on the position of nitrogen in the heterocyclic compounds, and the ability to bind Aβ was shown to be reduced when introducing anionic substituents on the thiophene backbone. Our findings provide the structural and functional basis for the development of ligands that can differentiate between aggregated proteinaceous species comprised of distinct proteins. These ligands might also be powerful tools for studying the pathogenesis of Aβ aggregation and for designing molecules for imaging of Aβ pathology.     

Amyloid Beta in Aging and Alzheimer’s Disease

Alzheimer’s disease (AD), is a progressive neurodegenerative disease that affects behavior, thinking, learning, and memory in elderly individuals. AD occurs in two forms, early onset familial and late-onset sporadic; genetic mutations in PS1, PS2, and APP genes cause early onset familial AD, and a combination of lifestyle, environment and genetic factors causes the late-onset sporadic form of the disease. However, accelerated disease progression is noticed in patients with familial AD. Disease-causing pathological changes are synaptic damage, and mitochondrial structural and functional changes, in addition to increased production and accumulation of phosphorylated tau (p-tau), and amyloid beta (Aβ) in the affected brain regions in AD patients. Aβ is a peptide derived from amyloid precursor protein (APP) by proteolytic cleavage of beta and gamma secretases. APP is a glycoprotein that plays a significant role in maintaining neuronal homeostasis like signaling, neuronal development, and intracellular transport. Aβ is reported to have both protective and toxic effects in neurons. The purpose of our article is to summarize recent developments of Aβ and its association with synapses, mitochondria, microglia, astrocytes, and its interaction with p-tau. Our article also covers the therapeutic strategies that reduce Aβ toxicities in disease progression and discusses the reasons for the failures of Aβ therapeutics.     

Thiophene-Based Dual Modulators of Aβ and Tau Aggregation

Alzheimer's disease is characterized by the accumulation of amyloid beta (Aβ) and Tau aggregates in the brain, which induces various pathological events resulting in neurodegeneration. There have been continuous efforts to develop modulators of the Aβ and Tau aggregation process to halt or modify disease progression. A few small-molecule-based inhibitors that target both Aβ and Tau pathology have been reported. Here, we report the screening of a targeted library of small molecules to modulate Aβ and Tau aggregation together with their in vitro, in silico and cellular studies. In vitro ThT fluorescence assay, dot blot assay, gel electrophoresis and transmission electron microscopy (TEM) results have shown that thiophene-based lead molecules effectively modulate Aβ aggregation and inhibit Tau aggregation. In silico studies performed by employing molecular docking, molecular dynamics and binding-free energy calculations have helped in understanding the mechanism of interaction of the lead thiophene compounds with Aβ and Tau fibril targets. In cellulo studies revealed that the lead candidate is biocompatible and effectively ameliorates neuronal cells from Aβ and Tau-mediated amyloid toxicity.     

Ac‐LPFFD‐Th: A Trehalose‐Conjugated Peptidomimetic as a Strong Suppressor of Amyloid‐β Oligomer Formation and Cytotoxicity

The inhibition of amyloid formation is a promising therapeutic approach for the treatment of neurodegenerative diseases. Peptide‐based inhibitors, which have been widely investigated, are generally derived from original amyloid sequences. Most interestingly, trehalose, a nonreducing disaccharide of α‐glucose, is effective in preventing the aggregation of numerous proteins. We have determined that the development of hybrid compounds could provide new molecules with improved properties that might synergically increase the potency of their single moieties. In this work, the ability of Ac‐LPFFD‐Th, a C‐terminally trehalose‐conjugated derivative, to slow down the Aβ aggregation process was investigated by means of different biophysical techniques, including thioflavin T fluorescence, dynamic light scattering, ESI‐MS, and NMR spectroscopy. Moreover, we demonstrate that Ac‐LPFFD‐Th modifies the aggregation features of Aβ and protects neurons from Aβ oligomers' toxic insult.     

Retro‐Enantio N‐Methylated Peptides as β‐Amyloid Aggregation Inhibitors

An emerging and attractive target for the treatment of Alzheimer's disease is to inhibit the aggregation of β‐amyloid protein (Aβ). We applied the retro‐enantio concept to design an N‐methylated peptidic inhibitor of the Aβ42 aggregation process. This inhibitor, inrD, as well as the corresponding all‐L (inL) and all‐D (inD) analogues were assayed for inhibition of Aβ42 aggregation. They were also screened in neuroblastoma cell cultures to assess their capacity to inhibit Aβ42 cytotoxicity and evaluated for proteolytic stability. The results reveal that inrD and inD inhibit Aβ42 aggregation more effectively than inL, that inrD decreases Aβ42 cytotoxicity to a greater extent than inL and inD, and that as expected, both inD and inrD are stable to proteases. Based on these results, we propose that the retro‐enantio approach should be considered in future designs of peptide inhibitors of protein aggregation.     

辅酶Q10对H_2O_2损伤PC12细胞保护作用的实验研究

研究通过H2O2诱导的PC12细胞建立阿尔茨海默症氧化损伤细胞模型,以细胞活性、丙二醛含量、谷胱甘肽含量及谷胱甘肽转移酶活性水平为指标,检测Co Q10对PC12细胞氧化应激损伤的保护作用。结果表明Co Q10能够提高H2O2所致PC12细胞的细胞活力的降低,降低了由H2O2诱导的脂质过氧化程度,并且可以抑制H2O2诱导的PC12细胞GSH含量及GST活性的降低。Co Q10对H2O2损伤的PC12细胞具有保护作用,可能是通过降低脂质过氧化程度且提高谷胱甘肽含量及谷胱甘肽转移酶活性实现的。     

Coarse-grained and All-atom Simulations towards the Early and Late Steps of Amyloid Fibril Formation

Alzheimer's disease is the most common neurodegenerative disease. Experiments and computer simulations can complement one another to provide a full and in-depth understanding of many aspects in the amyloid field at the atomistic level. Here, we review results of our coarse-grained and all-atom simulations in aqueous solution aimed at determining: 1) early aggregation steps of short linear peptides; 2) nucleation size number; 3) solution structure of the Aβ_1–40/Aβ_1–42 wild-type dimers; 4) impact of FAD (familial forms of Alzheimer's disease) mutations on the structure of Aβ_1–40/Aβ_1–42 dimers; and 5) impact of protective mutations on the structure of Aβ_1–40/Aβ_1–42 dimers.     

Atomistic characterization of binding modes and affinity of peptide inhibitors to amyloid-β protein

The aggregation of amyloid β-protein (Aβ) is tightly linked to the pathogenesis of Alzheimer’s disease. Previous studies have found that three peptide inhibitors (i.e., KLVFF, VVIA, and LPFFD) can inhibit Aβ aggregation and alleviate Aβ-induced neurotoxicity. However, atomic details of binding modes and binding affinities between these peptide inhibitors and Aβ have not been revealed. Here, using molecular dynamics simulations and molecular mechanics Poisson Boltzmann surface area (MM/PBSA) analysis, we examined the effect of three peptide inhibitors (KLVFF, VVIA, and LPFFD) on their sequence-specific interactions with Aβ and the molecular basis of their inhibition. All inhibitors exhibit varied binding affinity to Aβ, in which KLVFF has the highest binding affinity, whereas LPFFD has the least. MM/PBSA analysis further revealed that different peptide inhibitors have different modes of interaction with Aβ, consequently hotspot binding residues, and underlying driving forces. Specific residue-based interactions between inhibitors and Aβ were determined and compared for illustrating different binding and inhibition mechanisms. This work provides structure-based binding information for further modification and optimization of these three peptide inhibitors to enhance their binding and inhibitory abilities against Aβ aggregation.     

Molecular Characteristics of Amyloid Precursor Protein (APP) and Its Effects in Cancer

Amyloid precursor protein (APP) is a type 1 transmembrane glycoprotein, and its homologs amyloid precursor-like protein 1 (APLP1) and amyloid precursor-like protein 2 (APLP2) are highly conserved in mammals. APP and APLP are known to be intimately involved in the pathogenesis and progression of Alzheimer’s disease and to play important roles in neuronal homeostasis and development and neural transmission. APP and APLP are also expressed in non-neuronal tissues and are overexpressed in cancer cells. Furthermore, research indicates they are involved in several cancers. In this review, we examine the biological characteristics of APP-related family members and their roles in cancer.     

Natural Products Targeting Amyloid Beta in Alzheimer’s Disease

Alzheimer’s disease (AD) is a neurodegenerative disease characterized by severe brain damage and dementia. There are currently few therapeutics to treat this disease, and they can only temporarily alleviate some of the symptoms. The pathogenesis of AD is mainly preceded by accumulation of abnormal amyloid beta (Aβ) aggregates, which are toxic to neurons. Therefore, modulation of the formation of these abnormal aggregates is strongly suggested as the most effective approach to treat AD. In particular, numerous studies on natural products associated with AD, aiming to downregulate Aβ peptides and suppress the formation of abnormal Aβ aggregates, thus reducing neural cell death, are being conducted. Generation of Aβ peptides can be prevented by targeting the secretases involved in Aβ-peptide formation (secretase-dependent). Additionally, blocking the intra- and intermolecular interactions of Aβ peptides can induce conformational changes in abnormal Aβ aggregates, whereby the toxicity can be ameliorated (structure-dependent). In this review, AD-associated natural products which can reduce the accumulation of Aβ peptides via secretase- or structure-dependent pathways, and the current clinical trial states of these products are discussed.     

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.