Abeta Oligomers Research Articles

Dynamic neuronal regulatory network during the progression of Alzheimer’s disease suggests an adaptive survival strategy

Alzheimer’s disease (AD) is the most common neurodegenerative disease. In this work, comprehensive analyses on transcriptome studies of human postmortem brain tissues from AD patients revealed stepwise breakdown of the cellular machinery during the progression of AD at semi-quantitative level. At the early stage of AD, the accumulation of A-beta oligomers and amyloid plaques leads to the down-regulation of biosynthesis and energy metabolism, likely a response to the reduced level of nutrient and oxygen supply. At the intermediate stage, the progression of the disease leads to enhanced signal transduction, also likely an adjustment to the deteriorating environment. The late stage is characterized by the elevated apoptosis due to the excessive regulatory and repair burden. Interestingly, the elevated apoptosis at the late stage is accompanied by elevated anti-apoptotic signals, suggesting an adaptive survival strategy even at the late stage of AD. These findings can serve as theoretical basis for pharmaceutical intervention of Alzheimer’s disease.

Natural Amyloid-Beta Oligomers Acutely Impair the Formation of a Contextual Fear Memory in Mice

Memory loss is one of the hallmark symptoms of Alzheimer's disease (AD). It has been proposed that soluble amyloid-beta (Abeta) oligomers acutely impair neuronal function and thereby memory. We here report that natural Abeta oligomers acutely impair contextual fear memory in mice. A natural Abeta oligomer solution containing Abeta monomers, dimers, trimers, and tetramers was derived from the conditioned medium of 7PA2 cells, a cell line that expresses human amyloid precursor protein containing the Val717Phe familial AD mutation. As a control we used 7PA2 conditioned medium from which Abeta oligomers were removed through immunodepletion. Separate groups of mice were injected with Abeta and control solutions through a cannula into the lateral brain ventricle, and subjected to fear conditioning using two tone-shock pairings. One day after fear conditioning, mice were tested for contextual fear memory and tone fear memory in separate retrieval trials. Three experiments were performed. For experiment 1, mice were injected three times: 1 hour before and 3 hours after fear conditioning, and 1 hour before context retrieval. For experiments 2 and 3, mice were injected a single time at 1 hour and 2 hours before fear conditioning respectively. In all three experiments there was no effect on tone fear memory. Injection of Abeta 1 hour before fear conditioning, but not 2 hours before fear conditioning, impaired the formation of a contextual fear memory. In future studies, the acute effect of natural Abeta oligomers on contextual fear memory can be used to identify potential mechanisms and treatments of AD associated memory loss.

Cellular Internalization of Monomeric and Oligomeric Amyloid-Beta 42 Peptide

Amyloid beta (Abeta) is the major component of extracellular plaques found in Alzheimer's disease (AD). Uptake of Abeta from extracellular to intracellular space, which is likely by endocytosis, appears to be an important process for understanding AD pathology (Friedrich et al., PNAS 2010; Hu et al., PNAS 2009). It may also be a promising target for treatment and prevention.We aimed to study biophysical details of Abeta uptake, and to determine the uptake kinetics for different Abeta forms: monomers, oligomers, higher molecular mass aggregates of Abeta in and small fibrils. We used neuroblastoma (Sh-EP) cells as a model for neurons. Uptakes kinetic were followed by using the fluorescent labelled Abeta 42 that was monomerized and was purified by gel filtration. Abeta 42 peptide was added to the cell culture medium and the amount of intracellular aggregates was quantified using automated fluorescence microscopy.We compared uptake kinetics and aggregation kinetics in buffer and in cell culture medium at different Abeta 42 concentrations to test whether aggregation precedes uptake or vice versa. To compare the uptake of different Abeta species, pre-aggregated Abeta oligomers or small fibrils were added to the cells. We found that pre-aggregation accelerated the formation of intracellular aggregates, which suggests that Abeta oligomers and / or small fibrils may be taken up more rapidly than Abeta monomer.In the future the form and location of intracellular Abeta will be monitored by high resolution fluorescence nanoscopy combined with atomic force microscopy.

CNS cholesterol metabolism is associated with soluble amyloid precursor protein production in Alzheimer's disease

induced by Abeta. There is compelling evidence that Cu and Zn bind directly to Abeta in AD. This formation of Cu/Zn-Abeta complexes is thought to be aberrant as they have been detected only in AD, but not under healthy conditions. In this context, the understanding of how these metal ions interact with Abeta, their influence on structure and oligomerisation becomes an important issue for AD. Moreover, the mechanism of ROS production by Cu-Abeta in relation to its aggregation state, as well as metal exchange reaction from and to Abeta are crucial in order to understand why Abeta oligomers are highly toxic and why Abeta binding to Cu and Zn was only observed in AD. We hypothized that proteins able to retrieve the aberrant metal-binding to Abeta under healthy conditions but fail in AD might be involved. Metallothionein-3 and serum albumin might play this role, as they are highly abundant and strongly bind metal ions. Methods: In vitro biophysical techniques and studies in neuroblastoma cell culture were employed to understand the interaction of two of the most abundant metal-binding proteins, metallothionein and serum albumin and their capability of rescue metal-Abeta induced neurotoxicity. Results: In vitro experiments show that metallothionein and serum albumin are able to withdraw Cu ions from soluble and aggregated Abeta and suppress the ROS production of Cu-Abeta. Studies in cell culture confirmed the protection of these proteins against Cu-Abeta induced ROS and cell death. Metallothionein was more efficient then serum albumin. Moreover, metallothionein is also able to transfer zinc(II) to Abeta which modulates the aggregation behavior. This is enhanced under oxidative stress conditions. Conclusions: The results suggests that metallothioneins and perhaps also serum albumin are native protectors against the aberrant metal-binding to Abeta and hence neurotoxicity. Under oxidative stress conditions this activity might be reduced and favors the development of AD.

Therapeutic Monoclonal Antibodies against Abeta oligomers for Treatment of Alzheimer's disease

Therapeutic Monoclonal Antibodies against Abeta oligomers for Treatment of Alzheimer's disease

Antibody therapy for Alzheimer's disease

In order to avoid Abeta-induced autoimmune encephalitis, several monoclonal and polyclonal antibodies are in clinical trials. These are bapineuzumab, solanezumab, ponezumab, gantenerumab, BAN2401, gammaguard and octagam. Since each antibody has a different antigen epitope of Abeta, anti-amyloid activities are different. It is unknown which antibody is effective for Alzheimer disease, and we must wait for the result of clinical trials. Some patients who developed tissue amyloid plaque immuno-reactive (TAPIR) antibody showed slower decline after AN-1792 vaccination. We developed TAPIR-like monoclonal antibody, which was found to react with Abeta oligomers preferentially.

Specificity and sensitivity of the Abeta oligomer ELISA

Abeta soluble oligomers are believed to play a key role in the development of Alzheimer's disease (AD). An enzyme-linked immunosorbent assay (ELISA) commonly used to measure these proteins uses the same monoclonal antibody as both capture and reporter antibody. The objective of this study was to examine the specificity and sensitivity of this procedure, using monoclonal anti-Abeta antibody 6E10 as capture antibody and biotinylated 6E10 as reporter antibody. At comparable concentrations of Abeta soluble oligomers and low molecular weight (LMW) Abeta peptides, optical density (OD) values were four- to five-fold higher for the oligomer preparation than for the LMW Abeta. The LMW Abeta preparation, when evaluated by western blots of gels run under native conditions, showed only one band even after storage at 4°C for more than two months, suggesting that the ELISA was detecting Abeta monomer as well as Abeta oligomers. Possible explanations for these results are that (1) the LMW Abeta preparation may contain Abeta oligomer species below the limit of detection of western blot, but still detectable by ELISA, or (2) some nonspecific binding of the LMW Abeta to the ELISA plate may have occurred, allowing its relevant epitope to remain available for binding by the reporter antibody. Because of the possibility that this ELISA may not be oligomer-specific, it seems prudent to suggest that it should be used in combination with other methods, rather than as the sole technique, for measuring Abeta oligomers in biological specimens.

Interaction between Human Prion Protein and Amyloid-β (Aβ) Oligomers: ROLE OF N-TERMINAL RESIDUES

Soluble oligomers of Abeta42 peptide are believed to play a major role in the pathogenesis of Alzheimer disease (AD). It was recently found that at least some of the neurotoxic effects of these oligomers may be mediated by specific binding to the prion protein, PrP(C), on the cell surface (Laurén, J., Gimbel, D. A., Nygaard, H. B., Gilbert, J. W., and Strittmatter, S. M. (2009) Nature 457, 1128-1132). Here we characterized the interaction between synthetic Abeta42 oligomers and the recombinant human prion protein (PrP) using two biophysical techniques: site-directed spin labeling and surface plasmon resonance. Our data indicate that this binding is highly specific for a particular conformation adopted by the peptide in soluble oligomeric species. The binding appears to be essentially identical for the Met(129) and Val(129) polymorphic forms of human PrP, suggesting that the role of PrP codon 129 polymorphism as a risk factor in AD is due to factors unrelated to the interaction with Abeta oligomers. It was also found that in addition to the previously identified approximately 95-110 segment, the second region of critical importance for the interaction with Abeta42 oligomers is a cluster of basic residues at the extreme N terminus of PrP (residues 23-27). The deletion of any of these segments results in a major loss of the binding function, indicating that these two regions likely act in concert to provide a high affinity binding site for Abeta42 oligomers. This insight may help explain the interplay between the postulated protective and pathogenic roles of PrP in AD and may contribute to the development of novel therapeutic strategies as well.

Prion protein and Aβ‐related synaptic toxicity impairment

Alzheimer's disease (AD), the most common neurodegenerative disorder, goes along with extracellular amyloid-β (Aβ) deposits. The cognitive decline observed during AD progression correlates with damaged spines, dendrites and synapses in hippocampus and cortex. Numerous studies have shown that Aβ oligomers, both synthetic and derived from cultures and AD brains, potently impair synaptic structure and functions. The cellular prion protein (PrPC) was proposed to mediate this effect. We report that ablation or overexpression of PrPC had no effect on the impairment of hippocampal synaptic plasticity in a transgenic model of AD. These findings challenge the role of PrPC as a mediator of Aβ toxicity.

Synergistic effects of long-term antioxidant diet and behavioral enrichment on beta-amyloid load and non-amyloidogenic processing in aged canines.

A long-term intervention (2.69 years) with an antioxidant diet, behavioral enrichment, or the combined treatment preserved and improved cognitive function in aged canines. Although each intervention alone provided cognitive benefits, the combination treatment was additive. We evaluate the hypothesis that antioxidants, enrichment, or the combination intervention reduces age-related beta-amyloid (Abeta) neuropathology, as one mechanism mediating observed functional improvements. Measures assessed were Abeta neuropathology in plaques, biochemically extractable Abeta(40) and Abeta(42) species, soluble oligomeric forms of Abeta, and various proteins in the beta-amyloid precursor protein (APP) processing pathway. The strongest and most consistent effects on Abeta pathology were observed in animals receiving the combined antioxidant and enrichment treatment. Specifically, Abeta plaque load was significantly decreased in several brain regions, soluble Abeta(42) was decreased selectively in the frontal cortex, and a trend for lower Abeta oligomer levels was found in the parietal cortex. Reductions in Abeta may be related to shifted APP processing toward the non-amyloidogenic pathway, because alpha-secretase enzymatic activity was increased in the absence of changes in beta-secretase activity. Although enrichment alone had no significant effects on Abeta, reduced Abeta load and plaque maturation occurred in animals receiving antioxidants as a component of treatment. Abeta measures did not correlate with cognitive performance on any of the six tasks assessed, suggesting that modulation of Abeta alone may be a relatively minor mechanism mediating cognitive benefits of the interventions. Overall, the data indicate that multidomain treatments may be a valuable intervention strategy to reduce neuropathology and improve cognitive function in humans.

Mechanistic Investigation of the Inhibition of Aβ42 Assembly and Neurotoxicity by Aβ42 C-Terminal Fragments

Oligomeric forms of amyloid beta-protein (Abeta) are key neurotoxins in Alzheimer's disease (AD). Previously, we found that C-terminal fragments (CTFs) of Abeta42 interfered with assembly of full-length Abeta42 and inhibited Abeta42-induced toxicity. To decipher the mechanism(s) by which CTFs affect Abeta42 assembly and neurotoxicity, here, we investigated the interaction between Abeta42 and CTFs using photoinduced cross-linking and dynamic light scattering. The results demonstrate that distinct parameters control CTF inhibition of Abeta42 assembly and Abeta42-induced toxicity. Inhibition of Abeta42-induced toxicity was found to correlate with stabilization of oligomers with a hydrodynamic radius (R(H)) of 8-12 nm and attenuation of formation of oligomers with an R(H) of 20-60 nm. In contrast, inhibition of Abeta42 paranucleus formation correlated with CTF solubility and the degree to which CTFs formed amyloid fibrils themselves but did not correlate with inhibition of Abeta42-induced toxicity. Our findings provide important insight into the mechanisms by which different CTFs inhibit the toxic effect of Abeta42 and suggest that stabilization of nontoxic Abeta42 oligomers is a promising strategy for designing inhibitors of Abeta42 neurotoxicity.

Cerebrospinal Fluid from Alzheimer's Disease Patients Promotes Amyloid β-Protein Oligomerization

Oligomers of the amyloid beta-protein (Abeta) play an important role in Alzheimer's disease (AD). We hypothesized that AD patients have a central nervous system environment that promotes Abeta oligomerization. We investigated the effect of cerebrospinal fluid (CSF) from 33 patients with AD and 33 age-matched, non-demented controls on oligomerization of Abeta1-40 and Abeta1-42 using the technique of photo-induced cross-linking of unmodified proteins. CSF inhibited oligomerization of both Abeta1-40 and Abeta1-42. This inhibitory effect was significantly weaker in AD patients than in non-demented controls. Our results indicate that AD patients have a CSF environment favorable for Abeta oligomerization.

Amyloid- Peptide Oligomers Disrupt Axonal Transport through an NMDA Receptor-Dependent Mechanism That Is Mediated by Glycogen Synthase Kinase 3 in Primary Cultured Hippocampal Neurons

Disruption of axonal transport is a hallmark of several neurodegenerative diseases, including Alzheimer's disease (AD). Even though defective transport is considered an early pathologic event, the mechanisms by which neurodegenerative insults impact transport are poorly understood. We show that soluble oligomers of the amyloid-beta peptide (AbetaOs), increasingly recognized as the proximal neurotoxins in AD pathology, induce disruption of organelle transport in primary hippocampal neurons in culture. Live imaging of fluorescent protein-tagged organelles revealed a marked decrease in axonal trafficking of dense-core vesicles and mitochondria in the presence of 0.5 microm AbetaOs. NMDA receptor (NMDAR) antagonists, including d-AP5, MK-801, and memantine, prevented the disruption of trafficking, thereby identifying signals for AbetaO action at the cell membrane. Significantly, both pharmacological inhibition of glycogen synthase kinase-3beta (GSK-3beta) and transfection of neurons with a kinase-dead form of GSK-3beta prevented the transport defect. Finally, we demonstrate by biochemical and immunocytochemical means that AbetaOs do not affect microtubule stability, indicating that disruption of transport involves a more subtle mechanism than microtubule destabilization, likely the dysregulation of intracellular signaling cascades. Results demonstrate that AbetaOs negatively impact axonal transport by a mechanism that is initiated by NMDARs and mediated by GSK-3beta and establish a new connection between toxic Abeta oligomers and AD pathology.

Abeta oligomers cause local missorting of endogenous tau into dendrites, tau phosphorylation, destruction of microtubules and spines

Abnormal changes and aggregation of amyloid-beta (Abeta) and Tau protein are two hallmarks of Alzheimer's Disease (AD), and according to the Abeta-cascade hypothesis, Abeta is considered toxic for neurons and Tau a downstream target of Abeta. We have investigated primary hippocampal neurons for early and subtle changes following exposure to Abeta oligomers. Initial events become evident by missorting of endogenous Tau into the soma and dendrites, in contrast to their axonal sorting in normal neurons. In the missorted dendritic regions there is a depletion of spines and spine-related proteins. Tau in these regions shows elevated phosphorylation at several sites that are diagnostic of AD-Tau (notably at the 12E8 epitope, pS262/pS356, and the AT8 epitope, pS202/pT205), and local elevation of relevant kinase activity (e.g. microtubule affinity regulating kinase (MARK)). These local effects occur without major global changes in Tau, tubulin, kinase levels or activities. Missorting affects not only Tau, but also other axonal markers such as neurofilaments, and correlates with a local decrease of microtubules. Similar early changes can be evoked by other cell stressors. We conclude that Abeta oligomers evoke responses that disrupt the axonal sorting machinery, allow endogenous Tau to enter dendrites and to destroy spines and microtubules locally. This is in analogy to the loss of spines and microtubules observed in AD.

Effects of the English (H6R) and Tottori (D7N) Familial Alzheimer Disease Mutations on Amyloid β-Protein Assembly and Toxicity

Mutations in the amyloid beta-protein (Abeta) precursor gene cause autosomal dominant Alzheimer disease in a number of kindreds. In two such kindreds, the English and the Tottori, the mutations produce amyloid beta-proteins containing amino acid substitutions, H6R and D7N, respectively, at the peptide N terminus. To elucidate the structural and biological effects of the mutations, we began by examining monomer conformational dynamics and oligomerization. Relative to their wild type homologues, and in both the Abeta40 and Abeta42 systems, the English and Tottori substitutions accelerated the kinetics of secondary structure change from statistical coil --> alpha/beta --> beta and produced oligomer size distributions skewed to higher order. This skewing was reflected in increases in average oligomer size, as measured using electron microscopy and atomic force microscopy. Stabilization of peptide oligomers using in situ chemical cross-linking allowed detailed study of their properties. Each substitution produced an oligomer that displayed substantial beta-strand (H6R) or alpha/beta (D7N) structure, in contrast to the predominately statistical coil structure of wild type Abeta oligomers. Mutant oligomers functioned as fibril seeds, and with efficiencies significantly higher than those of their wild type homologues. Importantly, the mutant forms of both native and chemically stabilized oligomers were significantly more toxic in assays of cell physiology and death. The results show that the English and Tottori mutations alter Abeta assembly at its earliest stages, monomer folding and oligomerization, and produce oligomers that are more toxic to cultured neuronal cells than are wild type oligomers.

Hypercholesterolemia accelerates memory impairment in a transgenic mouse model of Abeta oligomers

Hypercholesterolemia accelerates memory impairment in a transgenic mouse model of Abeta oligomers

Synaptotoxicity of Abeta oligomers results from “hijacking” of metabotropic glutamate receptors

Synaptotoxicity of Abeta oligomers results from “hijacking” of metabotropic glutamate receptors

Detection of Abeta oligomers in Alzheimer's disease patient CSF with a novel misfolded protein assay

Detection of Abeta oligomers in Alzheimer's disease patient CSF with a novel misfolded protein assay

Globular state in the oligomers formed by Abeta peptides.

Replica exchange molecular dynamics and implicit solvent model are used to study two oligomeric species of Abeta peptides, dimer and tetramer, which are typically observed in in vitro experiments. Based on the analysis of free energy landscapes, density distributions, and chain flexibility, we propose that the oligomer formation is a continuous transition occurring without metastable states. The density distribution computations suggest that Abeta oligomer consists of two volume regions-the core with fairly flat density profile and the surface layer with rapidly decreasing density. The core is mostly formed by the N-terminal residues, whereas the C-terminal tends to occur in the surface layer. Lowering the temperature results in the redistribution of peptide atoms from the surface layer into the core. Using these findings, we argue that Abeta oligomer resembles polymer globule in poor solvent. Abeta dimers and tetramers are found to be structurally similar suggesting that the conformations of Abeta peptides do not depend on the order of small oligomers.

Higher Soluble Amyloid β Concentration in Frontal Cortex of Young Adults than in Normal Elderly or Alzheimer's Disease

Little is known about the relationship between soluble amyloid beta (Abeta) and age. We have measured soluble and insoluble Abeta by enzyme-linked immunosorbent assay (ELISA) in post-mortem frontal cortex in normal brains (16-95 years) and AD. Insoluble Abeta increased with age, and was significantly higher in Alzheimer's disease (AD) than age-matched controls. However, levels of soluble Abeta declined with age and were significantly greater in younger adults than older adults with or without AD. In AD, insoluble : soluble Abeta ratio was much higher than in age-matched controls. The high levels of soluble Abeta in young adults included oligomeric species of Abeta(1-42). These observations do not preclude Abeta oligomers as neurotoxic mediators of AD but suggest that if they are, the toxicity may be restricted to certain species (eg, beta-pleated protofibrillar species not detected by our assay) or takes decades to manifest. The dramatically increased insoluble : soluble Abeta in AD points to an altered dynamic equilibrium of Abeta in AD, reflecting both enhanced aggregation and continued overproduction or impaired removal of the soluble peptide in older age, when the concentration of this peptide should be declining.