Amyloid-derived Diffusible Ligands Research Articles

Preparation and qualification of soluble amyloid beta oligomers for use in bioanalytic assays supporting Alzheimer’s disease therapeutics

AbstractBackgroundSoluble amyloid beta oligomers (sAβOs) accumulate early in Alzheimer’s disease (AD) and trigger AD‐related neuropathologies and impairments in memory. sAβO structures contributing to the neurotoxic effects remain ill‐defined due to their low concentration, instability, and heterogeneity, impeding the effective design and use of sAβO reference standards in bioanalytic assays for use in clinical trials. At present, no assays for sAβOs are available with proven robustness and clinical performance, due to absence of well‐characterized critical raw materials (antibodies, reference materials for preparation of sAβO calibrators) and quality control specimens.MethodWe have used amyloid‐derived diffusible ligands (ADDLs), a synthetic model preparation commonly used in the literature (Stine et al. 2003), as a sAβO standard integrated into different assays designs developed on the Mesoscale Discovery (MSD) technology. As proof‐of‐concept, we have utilized these ADDL assays to study the specificity and selectivity of antibodies targeted against sAβOs.ResultOptimization of ADDL coating, stabilization of coated materials, and long‐term plate stability indicated that ADDL‐coated plates provided a suitable platform to assess the performance of sAβO‐targeting antibodies.Prior to coating, ADDL preparations were tested for reproducible SDS‐stable size distribution and immunoreactivity using SDS‐PAGE with silver stain and Western immunoblotting. The native size distribution (SEC) and globular conformation (AFM) of ADDLs and their ability to bind neuronal synapses in culture (ICC) and subsequently elicit AD‐related pathologies has been well reported in the literature. ADDLs were coated onto 96‐well MSD plates and the ADDL‐coated plates were stored at 4 °C for long‐term use; plate stability was confirmed up to one year. Biotinylated and non‐biotinylated sAβO‐targeting antibodies with variable selectivity towards monomeric or oligomeric Aβ species were used for comparative binding analysis. Additional sAβO preparations, including low concentration ADDL preparations and commercially available sAβOs were used to further understand the selectivity and specificity of the panel of sAβO‐targeting antibodies.ConclusionQualified sAβO preparations (ADDLs) can be used as tools for coating plates to determine specificity and selectivity of sAβO‐targeting antibodies. Other expected uses are: (i) calibrator in immunoassays; or (ii) to screen for the presence of sAβO auto‐antibodies in biological fluids.

Characterization of the influence of Amyloid β (1-42) By Way of Modeling Synaptic Cleft with an RC Electronic Circuit

The research toward understanding the role of oligomeric amyloids in Alzheimer disease (AD) has gained a considerable attention in recent years. The information obtained in this direction showed that Amyloid Derived Diffusible Ligands (ADDLs) cause an ambiguous ions movements across lipid bilayers in plasma membrane and consequently alter its dielectric properties such as permeability. Nevertheless, how these amyloid protein selectively alters the permeability of cell membrane has not been noted down because none of specific receptor have been identified for ADDLs binding. As a result of the increase of cellular calcium level, local micromechanical properties of cells can change and thus induce reorganization of cytoskeletal structures that can cause neurodegenerative disorders such as AD. Therefore, in this study we have tried to specify the effect of alteration of permittivity and permeability of synaptic channel as modelling synaptic cleft by an RC circuit. By linking the electronic neuronal models that we have previously designed with the RC cleft model, when the information transferred from one neuron to another, the rate of error measured on the later neuron circuit with reference of the former one was determined with respect to the rate of change of R and C values individually. It was observed that C and R, and hence permeability of synaptic channel does effect the communication error. It is concluded that a healthy synaptic channel conserves optimum resistivity and capacity levels at which the inter-neuronal signalling is achieved at a minimum error. This results could be linked to hypothesizes that amyloids have a common feature in structuring channel-like concentrations which supported by their electrical activity as well as the rate of permeability of Calcium like cautions.

Amyloid Beta-Derived Diffusible Ligands (ADDLs) Induce Abnormal Expression of Insulin Receptors in Rat Hippocampal Neurons

Amyloid beta (Aβ) is an important pathogenic factor in Alzheimer's disease (AD). In this study, we investigated the hypothesis that administration of amyloid-derived diffusible ligands (ADDLs) prepared from a synthetic Aβ(1-42) amyloid peptide can cause defective expression of insulin receptors (IRs). To this end, primary rat hippocampal neurons were treated with various concentrations of ADDLs and expression levels of IRs were measured using real-time PCR and western blots. In these experiments, the expression of IRs significantly increased following treatment with low concentrations of Aβ(1-42). In contrast, when higher concentrations of Aβ(1-42) were applied, the number of apoptotic cells present increased, and expression of IRs significantly decreased. In combination, these results suggest that ADDLs is able to induce abnormal expression of IRs and interrupt normal insulin signaling, thereby potentially contributing to central insulin resistance that can occur during progression of AD.

Synthesis, Characterization and Potential Applications of Multifunctional PEO-PPOPEO- Magnetic Drug Delivery System

Magnetic nanocomposites (MNCs) have highly been acknowledged in the diagnostics and therapeutic applications. Particularly, the multifunctional MNCs have brought a variety of possibilities in targeted drug delivery as well as non-invasive multimodality imaging. A temperature-responsive magnetic drug delivery system has been developed which is made up of superparamagnetic iron oxide nanoparticles (SIONPs) core and Pluronic shell. The magnetic cores composed of congo red conjugated to SIONPs have been proved beneficial as multimodal imaging agents, while superparamagnetic properties facile conducting the nano or micro systems to the vicinity of targeted tissue. Polymer shell formed by stimuli-responsive Pluronic F127 poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) PEO-PPO-PEO block copolymer serves as the carrier for both hydrophilic and hydrophobic drugs. X-ray diffraction (XRD) and high-resolution transmission electron microscope (HR-TEM) were used to characterize as-synthesized MNCs. Furthermore, vibrating magnetometer experiments showed MNCs having a higher magnetization value than bare magnetic nanoparticles (MNPs) and easy to conduct with an external magnetic field. The hydrodynamic size of MNCs was found to be varying in response to the stimuli temperature. Once the temperature increased, the hydrodynamic radius of MNCs decreased. In addition, the feasibility of the system as a targeted drug delivery system for Alzheimer's diseases (AD) diagnosis and therapy was studied. Searching for reliable targeting molecule, recent approaches for identification of amyloid-β (Aβ) and its derivatives have been evaluated. Consequently, the amyloid-derived diffusible ligands antibodies (anti-ADDLs) have been nominated as potential targeting molecules which can be attached to the MNCs system. The possibility of anti-ADDLs conjugation to DDS has been found promising for the multifunctional drug delivery system for AD diagnosis and therapy. However, further experimental studies are required to assess the performance of the proposed DDS.

Rationally Designed Turn Promoting Mutation in the Amyloid-β Peptide Sequence Stabilizes Oligomers in Solution

Enhanced production of a 42-residue beta amyloid peptide (Aβ42) in affected parts of the brain has been suggested to be the main causative factor for the development of Alzheimer's Disease (AD). The severity of the disease depends not only on the amount of the peptide but also its conformational transition leading to the formation of oligomeric amyloid-derived diffusible ligands (ADDLs) in the brain of AD patients. Despite being significant to the understanding of AD mechanism, no atomic-resolution structures are available for these species due to the evanescent nature of ADDLs that hinders most structural biophysical investigations. Based on our molecular modeling and computational studies, we have designed Met35Nle and G37p mutations in the Aβ42 peptide (Aβ42Nle35p37) that appear to organize Aβ42 into stable oligomers. 2D NMR on the Aβ42Nle35p37 peptide revealed the occurrence of two β-turns in the V24-N27 and V36-V39 stretches that could be the possible cause for the oligomer stability. We did not observe corresponding NOEs for the V24-N27 turn in the Aβ21–43Nle35p37 fragment suggesting the need for the longer length amyloid peptide to form the stable oligomer promoting conformation. Because of the presence of two turns in the mutant peptide which were absent in solid state NMR structures for the fibrils, we propose, fibril formation might be hindered. The biophysical information obtained in this work could aid in the development of structural models for toxic oligomer formation that could facilitate the development of therapeutic approaches to AD.

Topical Review: Metallic Nanoparticles Array for Immunoassay

Noble metal particles, especially gold (Au) and silver (Ag) nanoparticles exhibit unique and tunable optical properties on account of their surface plasmon resonance (SPR). It can be widely used in the field of immunoassay. In this paper, we reviewed the design, fabrication methods and applications of the nanoparticles. Discrete dipole approximation (DDA) and finite difference time domain (FDTD) methods applied brute-force computational methods for calculation and analysis of optical properties of different nanoparticles. Current nanotechnologies such as focused ion beam (FIB), nanosphere lithography (NSL), and laser interference lithography (LIL) make it possible to fabricate nanoparticles with arbitrary shapes. These nanoparticles are used to detect binding signals excited from biosamples for immunoassay such as antibody and antigen, amyloid-derived diffusible ligands (ADDLs)-potential Alzheimer's disease (AD) biomarkers, and Staphylococcal enterotixn B (SEB) in the concentration of nano-Moore per liter (nM) level. Finally, a biochip with multi-channel detection of biotin-streptavidin based on localized surface plasmon resonance was introduced. Copyright © 2010 American Scientific Publishers

Amyloid-derived diffusible ligands acutely impair spatial memory and hippocampal insulin signaling

The exact mechanism underlying cognitive deficits associated with AD remains unknown; however, both elevated levels of amyloid-derived diffusible ligands (ADDLs) and impaired brain insulin signalling are associated with AD, and ADDLs have been shown in vitro to impair hippocampal insulin signalling. We recently demonstrated that intact hippocampal insulin signalling is required for optimal cognitive functioning; hence, we hypothesized that ADDLs may directly impair memory by attenuating hippocampal insulin signalling. Rats were given either ADDLS or vehicle to the left hippocampus 10 min prior to testing in a four-arm maze using a spatial memory (spontaneous alternation) task. In vivo microdialysis was used to examine the impact of ADDL adminstration on local hippocampal metabolism, and post mortem analyses of insulin signalling were performed. ADDL administration significantly impaired memory performance, GluT translocation, and insulin signalling. ADDLs act rapidly to downregulate hippocampal insulin signalling, leading directly to impaired cognitive performance. These data demonstrate, in vivo, acute amyloid-induced cognitive impairment and support the hypothesis that such impairment is via downregulation of hippocampal insulin signalling.

Optical Properties and Immunoassay Applications of Noble Metal Nanoparticles

Noble metal, especially gold (Au) and silver (Ag) nanoparticles exhibit unique and tunable optical properties on account of their surface plasmon resonance (SPR). In this paper, we mainly discussed the theory background of the enhanced optical properties of noble metal nanoparticles. Mie theory, transfer matrix method, discrete dipole approximation (DDA) method, and finite‐difference time domain (FDTD) method applied brute‐force computational methods for different nanoparticles optical properties. Some important nanostructure fabrication technologies such as nanosphere lithography (NSL) and focused ion beam (FIB) are also introduced in this paper. Moreover, these fabricated nanostructures are used in the plasmonic sensing fields. The binding signal between the antibody and antigen, amyloid‐derived diffusible ligands (ADDLs)‐potential Alzheimer′s disease (AD) biomarkers, and staphylococcal enterotixn B (SEB) in nano‐Moore per liter (nM) concentration level are detected by our designed nanobiosensor. They have many potential applications in the biosensor, environment protection, food security, and medicine safety for health, and so forth, fields.

Gold Nanoparticle Ring and Hole Structures for Sensing Proteins and Antigen–Antibody Interactions

A new experimentally simple nanosphere lithography method was used to fabricate gold ring and nanohole structures. The method is based on the simultaneous self-assembly of polystyrene microspheres and gold colloids in multilayers, by a vertical deposition method. Dissolution of polystyrene microspheres resulted in the formation of a monolayer, where holes are surrounded by gold nanoparticles. The dependency of the sensitivity of the sensor platform on the size of the holes and their density has been demonstrated. Furthermore, sensing experiments have shown a high sensitivity of the hole structure toward fibrinogen, amyloid-derived diffusible ligands, and a plant protein (AT5G07010.1). It was found that the position and shape of the localized surface plasmon resonance band changed significantly as a result of the antigen–antibody recognition event.

Influence of Cr Adhesion Layer on Detection of Amyloid-Derived Diffusible Ligands Based on Localized Surface Plasmon Resonance

A Cr adhesion layer inserted between Ag nanoparticles and a glass substrate, for the purpose of improving the adhesion of Ag nanoparticles to glass, was observed to cause an abnormal peak shift of extinction spectra in non-specific reactions. The undesired peak shift misleads molecule detection in non-specific reactions. To solve this issue, a practical technique using n-propyl-trimethoxysilane-based passivation for the detection of amyloid-derived diffusible ligands was investigated as a route to eliminate the abnormal peak shifting observed in the non-specific reactions. To evaluate this passivation technique, localized surface plasmon resonance immunoassay experiments were conducted. Experimental results derived with and without the passivation process were investigated as a basis for comparative analysis. Our experimental results demonstrate that this passivation technique effectively eliminates the observed peak shift originating from the Cr adhesion layer.

Emerging concepts of how β-amyloid proteins and pro-inflammatory cytokines might collaborate to produce an ‘Alzheimer brain’ (Review)

Three steps lead to the development of full-blown sporadic or late-onset Alzheimer's disease or dementia (AD). In the young brain, amyloid β-(1-42) (Aβ 42) is a normal aggregation-prone protein product of neuronal activity that is kept at a safe low level by proteolysis in neurons and glial cells, and by expulsion across the blood-brain barrier. But clearance declines with advancing age. Step 1: Because of the normal decline with age of the Aβ 42-clearing mechanisms, toxic amyloid-derived diffusible ligands (ADDLs) made of dodecamers of the aggregation-prone Aβ 42 start accumulating. These Aβ 42 dodecamers selectively target the initially huge numbers of excitatory synapses of neurons and cause them to start slowly dropping, which increasingly impairs plasticity and sooner or later starts noticeably affecting memory formation. At a certain point, this increasing loss of synapses induces the neurons to redirect their still-expressed cell cycle proteins from post-mitotic jobs, such as maintaining synapses, to starting a cell cycle and partially or completely replicating DNA without entering mitosis. The resulting aneuploid or tetraploid neurons survive for as long as 6-12 months as quasi-functional 'undead zombies', with developing tangles of hyperphosphorylated τ protein disrupting the vital anterograde axonal transport of mitochondria and other synapse-vital components. Step 2: The hallmark AD plaques appear as Aβ 42 clearance continues to decline and the formation of Aβ 42 non-diffusible fibrils begins in the aging brain. Step 3: A terminal cytokine-driven maëlstrom begins in the aging brain when microglia, the brain's professional macrophages, are activated in and around the plaques. They produce pro-inflammatory cytokines, such as IFN-γ, IL-1β and TNF-α. One of these, IFN-γ, causes the astrocytes enwrapping the neuronal synapses to express their β-secretase (BACE1) genes and produce and release Aβ 42, which can kill the closely apposed neurons by binding to their p75NTR receptors, which generate apoptogenic signals. Astrocytes are also stimulated by the same cytokines to turn on their nitric oxide synthase (NOS)-2 genes and start pouring large amounts of nitric oxide (NO) and its cytocidal derivative peroxynitrite (ONOO-) directly out onto the closely apposed neurons.

Solution State Characterization of Amyloid β-Derived Diffusible Ligands

A growing body of evidence suggests that soluble oligomeric forms of the amyloid beta peptide known as amyloid-derived diffusible ligands (ADDLs) are the toxic species responsible for neurodegeneration associated with Alzheimer's disease. Accurate biophysical characterization of ADDL preparations is hampered by the peptide's strong tendency to self-associate and the effect of factors such as ionic strength, temperature, and pH on its behavior. In addition, amyloid peptides are known to interact with common laboratory excipients, specifically detergents, further complicating the results from standard analytical methods such as denaturing polyacrylamide gel electrophoresis. We have studied the solution behavior of various amyloid peptide preparations using analytical ultracentrifugation and size exclusion chromatography coupled with multiangle laser light scattering. Our results indicate that ADDL preparations exist in solution primarily as a binary mixture of a monomeric peptide and high-molecular mass oligomers. We relate our findings to previously described characterizations utilizing atomic force microscopy and electrophoretic methods and demonstrate that low-molecular mass oligomers identified by gel electrophoresis likely represent artifacts induced by the peptide's interaction with detergent, while atomic force microscopy results are likely skewed by differential binding of monomeric and oligomeric peptide species. Finally, we confirm that only the high-molecular mass oligomeric components of an ADDL preparation are capable of binding to subpopulations of primary hippocampal neurons in vitro.

PGH2‐derived levuglandin adducts increase the neurotoxicity of amyloid β1–42

The body of evidence indicating that oligomers of amyloid beta(1-42) (Abeta(1-42)) produce toxicity to neurons, together with our demonstration that prostaglandin H(2) (PGH(2)) oligomerizes amyloid beta(1-42), led to the examination of the neurotoxicity of amyloid beta(1-42) treated with PGH(2). The neurotoxic effects of Abeta(1-42) incubated with PGH(2) was examined in primary cultures of cerebral neurons of mice, monitoring the reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as an indicator of cell toxicity. Whereas Abeta(1-42) itself, incubated for 24 h, has little or no effect on MTT reduction, Abeta(1-42) 24 h after exposure to PGH(2) produced a marked inhibition of MTT reduction, comparable with the inhibition resulting from Abeta(1-42) that has been oligomerized by incubation for 6 days. Similar results were obtained when Abeta(1-42) was incubated with levuglandin E(2) (LGE(2)), a reactive aldehyde formed by spontaneous rearrangement of PGH(2). The oligomers formed from reaction of Abeta(1-42) with LGE(2) exhibit immunochemical similarity with amyloid-derived diffusible ligands (ADDLs), as determined by analysis of the products of reaction of Abeta(1-42) with LGE(2) using western blotting with an antibody that is selective for ADDLs.

Identification of naturally occurring spirostenols preventing β-amyloid-induced neurotoxicity

22 R-Hydroxycholesterol is an intermediate in the steroid biosynthesis pathway shown to exhibit a neuroprotective property against β-amyloid (1–42) (Aβ) toxicity in rat PCl2 and human NT2N neuronal cells by binding and inactivating Aβ. In search of potent 22 R-hydroxycholesterol derivatives, we assessed the ability of a series of naturally occurring entities containing the 22 R-hydroxycholesterol structure to protect PC12 cells against Aβ-induced neurotoxicity, determined by measuring changes in membrane potential, mitochondrial diaphorase activity, ATP levels and trypan blue uptake. 22 R-Hydroxycholesterol derivatives sharing a common spirost-5-en-3-ol or a furost-5-en-3-ol structure were tested. Although some of these compounds were neuroprotective against 0.1 μM Aβ, only three protected against the 1–10 μM Aβ-induced toxicity and, in contrast to 22 R-hydroxycholesterol, all were devoid of steroidogenic activity. These entities shared a common structural feature, a long chain ester in position 3 and common stereochemistry. The neuroprotective property of these compounds was coupled to their ability to displace radiolabeled 22R-hydroxycholesterol from Aβ, suggesting that the Aβ-22 R-hydroxycholesterol physicochemical interaction contributes to their beneficial effect. In addition, a 22 R-hydroxycholesterol derivative inhibited the formation of neurotoxic amyloid-derived diffusible ligands. Computational docking simulations of 22 R-hydroxycholesterol and its derivatives on Aβ identified two binding sites. Chemical entities, as 22 R-hydroxycholesterol, seem to bind preferentially only to one site. In contrast, the presence of the ester chain seems to confer the ability to bind to both sites on Aβ, leading to neuroprotection against high concentrations of Aβ. In conclusion, these results suggest that spirost-5-en-3-ol naturally occurring derivatives of 22 R-hydroxycholesterol might offer a new approach for Alzheimer’s disease therapy.