Soluble Aβ Oligomers Research Articles

Anti-amyloid Therapy of Alzheimer's Disease: Current State and Prospects.

Drug development for the treatment of Alzheimer's disease (AD) has been for a long time focused on agents that were expected to support endogenous β-amyloid (Aβ) in a monomeric state and destroy soluble Aβ oligomers and insoluble Aβ aggregates. However, this strategy has failed over the last 20 years and was eventually abandoned. In this review, we propose a new approach to the anti-amyloid AD therapy based on the latest achievements in understanding molecular causes of cerebral amyloidosis in AD animal models.

Shear-Induced Amyloid Formation in the Brain: III. The Roles of Shear Energy and Seeding in a Proposed Shear Model.

If cerebrospinal and interstitial fluids move through very narrow brain flow channels, these restrictive surroundings generate varying levels of fluid shear and different shear rates, and dissolved amyloid monomers absorb different shear energies. It is proposed that dissolved amyloid-β protein (Aβ) and other amyloid monomers undergo shear-induced conformational changes that ultimately lead to amyloid monomer aggregation even at very low brain flow and shear rates. Soluble Aβ oligomers taken from diseased brains initiate in vivo amyloid formation in non-diseased brains. The brain environment is apparently responsible for this result. A mechanism involving extensional shear is proposed for the formation of a seed Aβ monomer molecule that ultimately promotes templated conformational change of other Aβ molecules. Under non-quiescent, non-equilibrium conditions, gentle extensional shear within the brain parenchyma, and perhaps even during laboratory preparation of Aβ samples, may be sufficient to cause subtle conformational changes in these monomers. These result from brain processes that significantly lower the high activation energy predicted for the quiescent Aβ dimerization process. It is further suggested that changes in brain location and changes brought about by aging expose Aβ molecules to different shear rates, total shear, and types of shear, resulting in different conformational changes in these molecules. The consequences of such changes caused by variable shear energy are proposed to underlie formation of amyloid strains causing different amyloid diseases. Amyloid researchers are urged to undertake studies with amyloids, anti-amyloid drugs, and antibodies while all of these are under shear conditions similar to those in the brain.

Soluble Aβ Oligomers Impair Dipolar Heterodendritic Plasticity by Activation of mGluR in the Hippocampal CA1 Region

SummarySoluble Aβ oligomers (oAβs) contribute importantly to synaptotoxicity in Alzheimer disease (AD), but the mechanisms related to heterogeneity of synaptic functions at local circuits remain elusive. Nearly all studies of the effects of oAβs on hippocampal synaptic plasticity have only examined homosynaptic plasticity. Here we stimulated the Schaffer collaterals and then simultaneously recorded in stratum radiatum (apical dendrites) and stratum oriens (basal dendrites) of CA1 neurons. We found that the apical dendrites are significantly more vulnerable to oAβ-mediated synaptic dysfunction: the heterosynaptic basal dendritic long-term potentiation (LTP) remained unchanged, whereas the homosynaptic apical LTP was impaired. However, the heterosynaptic basal dendritic plasticity induced by either spaced 10-Hz bursts or low-frequency (1-Hz) stimulation was disrupted by oAβs in a mGluR5-dependent manner. These results suggest that different firing patterns in the same neurons may be selectively altered by soluble oAβs in an early phase of AD, before frank neurodegeneration.

Effects of Prion Protein on Aβ42 and Pyroglutamate-Modified AβpΕ3-42 Oligomerization and Toxicity.

Soluble Aβ oligomers are widely recognized as the toxic forms responsible for triggering AD, and Aβ receptors are hypothesized to represent the first step in a neuronal cascade leading to dementia. Cellular prion protein (PrP) has been reported as a high-affinity binder of Aβ oligomers. The interactions of PrP with both Aβ42 and the highly toxic N-truncated pyroglutamylated species (AβpE3-42) are here investigated, at a molecular level, by means of ThT fluorescence, NMR and TEM. We demonstrate that soluble PrP binds both Aβ42 and AβpE3-42, preferentially interacting with oligomeric species and delaying fibril formation. Residue level analysis of Aβ42 oligomerization process reveals, for the first time, that PrP is able to differently interact with the forming oligomers, depending on the aggregation state of the starting Aβ42 sample. A distinct behavior is observed for Aβ42 1-30 region and C-terminal residues, suggesting that PrP protects Aβ42 N-tail from entangling on the mature NMR-invisible fibril, consistent with the hypothesis that Aβ42 N-tail is the locus of interaction with PrP. PrP/AβpE3-42 interactions are here reported for the first time. All interaction data are validated and complemented by cellular tests performed on Wt and PrP-silenced neuronal cell lines, clearly showing PrP dependent Aβ oligomer cell internalization and toxicity. The ability of soluble PrP to compete with membrane-anchored PrP for binding to Aβ oligomers bears relevance for studies of druggable pathways.

Alzheimer’s disease as a metabolic disorder

Alzheimer’s disease (AD) is defined by memory loss and cognitive impairment, along with the accumulation in brain of two types of abnormal structures, extracellular amyloid plaques and intraneuronal neurofibrillary tangles. Both plaques and tangles are composed predominantly of poorly soluble filaments that respectively assemble from amyloid-β (Aβ) peptides and the neuron-specific, microtubule-associated protein, tau. It is now widely acknowledged that soluble oligomers of Aβ and tau, the building blocks of plaques and tangles, are principal drivers of AD pathogenesis by acting coordinately to impair and destroy synapses, and kill neurons. The behavioral features of AD are a direct consequence of these attacks on synapses and neuronal viability, which in turn reflect a reduced capacity of AD neurons to utilize energy sources needed to maintain neuronal function and vitality. In other words, AD neurons are starving, even when they may be surrounded by abundant nutrients. Here, we review some of the evidence for the metabolic deficiencies of neurons in AD and how they impact neuronal health.

OLIGOMERIC Aβ AS A MECHANISTIC BIOMARKER FOR ALZHEIMER’S DISEASE IN HUMAN PLASMA

APOE4 is the greatest genetic risk factor for Alzheimer's disease (AD), increasing risk up to 15-fold compared to the more common APOE3. Autosomal dominant mutations that increase the peptide amyloid-β (Aβ42) cause the rare familial form of AD. Aβ42 aggregate to form amyloid plaques and soluble oligomeric Aβ (oAβ), the latter considered a proximal neurotoxin. Among APOE4 carriers, females have an increased AD risk, rate of cognitive loss and accumulation of Aβ42 compared to males. Thus, our hypothesis is that oAβ is a mechanistic biomarker that can track AD progression based on APOE genotype and sex. Many current AD biomarkers for are not predictive, reliably diagnostic or easily measured, although CSF Aβ42 levels are considered a “gold standard.” To test the value of oAβ as a biomarker, we developed a unique monoclonal antibody, MOAB-2, that enabled development of an ELISA that detects 1pg/ml oAβ. ELISAs were run to determine oAβ levels in plasma from healthy controls and AD patients with APOE3 or APOE4 genotypes. In both human brain and CSF, oAβ and Aβ42 levels are higher in AD vs. control, but in the AD cohort, only oAβ is higher with APOE4 vs. APOE3, supporting our hypothesis that APOE-modulated changes in oAβ levels underlie the APOE4-induced risk for AD. Therefore, we measured oAβ in human plasma, which demonstrated that oAβ levels are higher in AD vs. control subjects. Stratification of the AD cohort by sex and APOE reveal significantly greater oAβ levels in APOE4 vs. APOE3 and females vs. males. Importantly, when the data is further stratified by sex within genotype, oAβ levels are higher in female APOE4 carriers vs. male APOE4 carriers. These results are consistent with the concept of personal medicine, as significance increases with stratification. This specifically supports the hypothesis that with subject stratification, plasma oAβ levels may become a new “gold standard.” Future longitudinal studies will determine thresholds and establish the predictive value of oAβ as a mechanistic biomarker. Such a mechanistic biomarker is critical for effective prediction of AD and will enable clinical studies to determine efficacy of predictive therapeutics.

Heme and hemoglobin suppress amyloid β–mediated inflammatory activation of mouse astrocytes

Glial immune activity is a key feature of Alzheimer's disease (AD). Given that the blood factors heme and hemoglobin (Hb) are both elevated in AD tissues and have immunomodulatory roles, here we sought to interrogate their roles in modulating β-amyloid (Aβ)-mediated inflammatory activation of astrocytes. We discovered that heme and Hb suppress immune activity of primary mouse astrocytes by reducing expression of several proinflammatory cytokines (e.g. RANTES (regulated on activation normal T cell expressed and secreted)) and the scavenger receptor CD36 and reducing internalization of Aβ(1-42) by astrocytes. Moreover, we found that certain soluble (>75-kDa) Aβ(1-42) oligomers are primarily responsible for astrocyte activation and that heme or Hb association with these oligomers reverses inflammation. We further found that heme up-regulates phosphoprotein signaling in the phosphoinositide 3-kinase (PI3K)/Akt pathway, which regulates a number of immune functions, including cytokine expression and phagocytosis. The findings in this work suggest that dysregulation of Hb and heme levels in AD brains may contribute to impaired amyloid clearance and that targeting heme homeostasis may reduce amyloid pathogenesis. Altogether, we propose heme as a critical molecular link between amyloid pathology and AD risk factors, such as aging, brain injury, and stroke, which increase Hb and heme levels in the brain.

Up-regulation of NSP3 by Oligomeric Aβ Accelerates Neuronal Death Through Cas-independent Rap1A Activation

β-Amyloid (Aβ) plays an important role in the early pathogenesis of Alzheimer’s disease (AD). In vitro studies have demonstrated that Aβ oligomers induce hippocampal and neocortical neuronal death. However the neurotoxic mechanisms by which soluble Aβ oligomers cause neuronal damage and death remain to be fully elucidated. To this end, we analyzed the gene expression profile of rat cerebral cortical neurons treated with Aβ oligomers in vitro. Aβ treatment induced the expression of novel SH2-containing protein 3 (NSP3), an adaptor molecule interacting with Cas family proteins. NSP3 expression was upregulated not only in oligomeric-Aβ-treated cultured neurons but also in the neocortex of aged Tg2576 mice. NSP3 overexpression in cultured cortical neurons accelerated neuronal death. The C-terminal region of NSP3 unbound to a Cas protein was necessary for the NSP3-induced acceleration of neuronal death, as was Cas-independent Rap1A activation downstream of NSP3. Moreover, NSP3 RNAi knockdown partially rescued Aβ-oligomer-treated neurons. These results indicate that NSP3 upregulation by soluble Aβ oligomers may accelerate neuronal death via Cas-independent Rap1A activation, implicating NSP3 in the pathogenesis of AD.

Improved synaptic and cognitive function in aged 3 × Tg-AD mice with reduced amyloid-β after immunotherapy with a novel recombinant 6Aβ15-TF chimeric vaccine

Alzheimer's disease (AD) is the most common progressive neurodegenerative disorder impairing memory and cognition. In this study, we describe the immunogenicity and protective efficacy of the novel recombinant 6Aβ15-TF chimeric antigen as a subunit protein vaccine for AD. Recombinant 6Aβ15-TF chimeric vaccine induced strong Aβ-specific humoral immune responses without Aβ-specific T cell immunity in C57/BL6 and 3 × Tg-AD mice at different ages. As an early immunotherapy model for AD, this vaccine induced high titers of long-lasting anti-Aβ42 antibodies in aged 3 × Tg-AD mice, which led to improve behavioral performance and markedly reduced the levels of insoluble and soluble Aβ and Aβ oligomers. In agreement with these findings, immunotherapy with 6Aβ15-TF prevented the Aβ-induced decrease of presynaptic and postsynaptic proteins in aged 3 × Tg-AD mice. Our results suggest that this novel and highly immunogenic recombinant 6Aβ15-TF chimeric vaccine provides neuroprotection in AD mice and can be considered an effective AD candidate vaccine.

Diffusible, highly bioactive oligomers represent a critical minority of soluble Aβ in Alzheimer's disease brain.

Significant data suggest that soluble Aβ oligomers play an important role in Alzheimer's disease (AD), but there is great confusion over what exactly constitutes an Aβ oligomer and which oligomers are toxic. Most studies have utilized synthetic Aβ peptides, but the relevance of these test tube experiments to the conditions that prevail in AD is uncertain. A few groups have studied Aβ extracted from human brain, but they employed vigorous tissue homogenization which is likely to release insoluble Aβ that was sequestered in plaques during life. Several studies have found such extracts to possess disease-relevant activity and considerable efforts are being made to purify and better understand the forms of Aβ therein. Here, we compared the abundance of Aβ in AD extracts prepared by traditional homogenization versus using a far gentler extraction, and assessed their bioactivity via real-time imaging of iPSC-derived human neurons plus the sensitive functional assay of long-term potentiation. Surprisingly, the amount of Aβ retrieved by gentle extraction constituted only a small portion of that released by traditional homogenization, but this readily diffusible fraction retained all of the Aβ-dependent neurotoxic activity. Thus, the bulk of Aβ extractable from AD brain was innocuous, and only the small portion that was aqueously diffusible caused toxicity. This unexpected finding predicts that generic anti-oligomer therapies, including Aβ antibodies now in trials, may be bound up by the large pool of inactive oligomers, whereas agents that specifically target the small pool of diffusible, bioactive Aβ would be more useful. Furthermore, our results indicate that efforts to purify and target toxic Aβ must employ assays of disease-relevant activity. The approaches described here should enable these efforts, and may assist the study of other disease-associated aggregation-prone proteins.

Porphyrin Cyclodextrin Conjugates Modulate Amyloid Beta Peptide Aggregation and Cytotoxicity.

Although fibrillar amyloid beta peptide (Aβ) aggregates are one of the major hallmarks of Alzheimer's disease, increasing evidence suggests that soluble Aβ oligomers are the primary toxic species. Targeting the oligomeric species could represent an effective strategy to interfere with Aβ toxicity. In this work, the biological properties of 5[4-(6-O-β-cyclodextrin)-phenyl],10,15,20-tri(4-hydroxyphenyl)-porphyrin and its zinc complex were tested, as new molecules that interact with Aβ and effectively prevent its cytotoxicity. We found that these systems can cross the cell membrane to deliver Aβ intracellularly and promote its clearance. Our results provide evidence for the use of cyclodextrin-porphyrin derivatives as a promising strategy to target amyloid aggregation.

A new purified Lawsoniaside remodels amyloid-β42 fibrillation into a less toxic and non-amyloidogenic pathway

Mounting evidence indicates soluble Aβ42 oligomers as the most toxic species causing neuronal death which leads to the onset and progression of Alzheimer disease (AD). Recently, it has been found that neurotoxic Aβ42 oligomers grow from monomeric species or arise following secondary nucleation by preformed mature fibrils. Thus, the use of natural compounds such as polyphenols to hinder the growth or to remodel Aβ42 fibrils is one of the most promising strategies for AD treatment. In our previous study, we showed that 1, 2, 4-trihydroxynaphthalene-2-O-β-d-glucopyranoside (THNG) inhibits Aβ42 aggregation during the early steps of the aggregation process, inhibits its conformational change to a β-sheet-rich structure, decreases its polymerization, inhibits its fibrillogenisis and reduces oxidative stress and aggregate cytotoxicity. Here, we used different spectroscopic and cell culture methods to check the effect of THNG on fibrils disaggregation. We showed that THNG binds to mature Aβ42 fibrils, rearrange their secondary structure, and remodels them into non-amyloid, less toxic, species by inhibiting their interaction with the plasma membrane. Our findings reveal that THNG is a good agent to remodel amyloid fibrils and could be used as a starting molecular scaffold to design new anti-AD drugs.

Effects of Single Amino Acid Substitutions on Aggregation and Cytotoxicity Properties of Amyloid β Peptide

Alzheimer’s disease is the main cause of dementia and the deposition of amyloid beta peptide (Aβ) in the brain is the key event in its progression. Soluble oligomers of Aβ are proposed to be the primary neurotoxic agents, and prevention of Aβ self-assembly has been proposed as a therapeutic approach. To analyze the role of key amino acids for Aβ aggregation and cytotoxicity, we introduced the three single mutations K28A, A30W or M35C in three length variants of Aβ: 25–35, 1–40, 1–42, 1–40. We assessed amyloid formation through atomic force microscopy and thioflavine fluorescence and tested the amyloid seeding effects of the mutant peptides in co-incubation assays. We also correlated changes in aggregation properties with cytotoxicity and reactive oxygen species production. Atomic force microscopy imaging demonstrated that the formation of amyloid fibrils was more dependent on the primary sequence of the peptides rather than on their length. We observe decreased formation of amyloid-like structures in all the three mutant Aβ (25–35) peptides, but these short peptide mutants remained cytotoxic. A30W and M35C mutants of the longer peptides decreased reactive oxygen species production and this effect was correlated with lower levels of cytotoxicity, but not with aggregation properties. Taken together, our results show that cytotoxicity of the Aβ peptide variants is more dependent on their primary amino acid sequence than on their capability to aggregate into amyloid-like structures.

Taxifolin prevents β-amyloid-induced impairments of synaptic formation and deficits of memory via the inhibition of cytosolic phospholipase A2/prostaglandin E2 content.

Taxifolin is a potent flavonoid with anti-inflammatory activity. Taxifolin has been reported to decrease the accumulation of β-amyloid (Aβ), and reduce Aβ-induced neurotoxicity. However, the detail molecular mechanism of taxifolin against Aβ-induced neurotoxicity is largely unknown. In this study, we revealed the protective effects and the underlying mechanisms of taxifolin on the impairments of cognitive function and synapse formation induced by soluble Aβ oligomers. Our results showed that taxifolin prevented neuronal cell death in a concentration-dependent manner. The recognition memory in novel object recognition tasks and the spatial memory in Morris water maze tests are significantly lower in the Alzheimer's disease (AD) model mice induced by hippocampal injection of Aβ42. Taxifolin treatment prevented the recognitive and spatial memory deficits of the AD mice. 10mg/kg taxifolin treatment also significantly prevented the decreased expression levels of PSD 95 induced by Aβ42. Live cell imaging study showed that 2h pre-treatment of taxifolin prevented the decrease in the number of filopodium and spine induced by Aβ42 oligomers. Aβ42 oligomers significantly increased the production of cytosolic phospholipase A2 (cPLA2), a crucial enzyme of pro-inflammatory mediator, and prostaglandin E2 (PGE2), a neuroinflammatory molecule. Taxifolin significantly reduced the content of cPLA2 and PGE2 induced by Aβ42 both in the primary hippocampal neurons and hippocampal tissues. These results indicated that taxifolin might prevent Aβ42 oligomer-induced synapse and cognitive impairments through decreasing cPLA2 and PGE2. Our study provided novel insights into the cellular mechanisms for the protective effects of taxifolin on AD.

Amyloid toxicity in Alzheimer’s disease

A major feature of Alzheimer's disease (AD) pathology is the plaque composed of aggregated amyloid-β (Aβ) peptide. Although these plaques may have harmful properties, there is much evidence to implicate soluble oligomeric Aβ as the primary noxious form. Aβ oligomers can be generated both extracellularly and intracellularly. Aβ is toxic to neurons in a myriad of ways. It can cause pore formation resulting in the leakage of ions, disruption of cellular calcium balance, and loss of membrane potential. It can promote apoptosis, cause synaptic loss, and disrupt the cytoskeleton. Current treatments for AD are limited and palliative. Much research and effort is being devoted to reducing Aβ production as an approach to slowing or preventing the development of AD. Aβ formation results from the amyloidogenic cleavage of human amyloid precursor protein (APP). Reconfiguring this process to disfavor amyloid generation might be possible through the reduction of APP or inhibition of enzymes that convert the precursor protein to amyloid.

Gas Phase Studies of the Amyloid-β Peptide

The amyloid-β (Aβ) peptide is an amphiphilic peptide that exhibits self-aggregating properties forming the amyloid fibrils that can be found in the brains of Alzheimer patients. Today it is believed that that it is the soluble Aβ oligomers rather than the mature fibrils that are the main neurotoxic species. These small peptide-assembles are known to associate with membranes and form pores that can transport Ca2+ into the cell, which in part could be responsible for their neurotoxic properties. However, most biophysical methods that have been developed to study Aβ target either the monomer or the mature fibrils, and not the low abundance and polydisperse oligomers.We have developed a soft ionization mass spectrometry (MS) method that retains and detects these non-covalent oligomer interactions in the gas phase. And since MS is a non-averaging technique the aggregation kinetics for different oligomeric species, as well as reduced and oxidized peptides, could be followed simultaneously. The high-resolution MS was also coupled to an ion mobility spectrometer that separates ions of the same m/z according to collisional cross section, yielding information about peptide conformations in the gas phase. Additionally, peptide interactions with other molecules, such as membrane mimicking detergents can be monitored. Large complexes between Aβ(1-40) and detergent micelles was shown to survive in the MS and could be identified. Detergent head group charges seem to be essential for peptide-micelle interactions, both in the gas phase and in solution, as no structure induction is observed upon addition of non-ionic detergent. The developed methodology is promising for future detection of pore-forming oligomers in micelles, and probably also in other more sophisticated membrane models.

Peptides as Potential Therapeutics for Alzheimer's Disease.

Intracellular synthesis, folding, trafficking and degradation of proteins are controlled and integrated by proteostasis. The frequency of protein misfolding disorders in the human population, e.g., in Alzheimer’s disease (AD), is increasing due to the aging population. AD treatment options are limited to symptomatic interventions that at best slow-down disease progression. The key biochemical change in AD is the excessive accumulation of per-se non-toxic and soluble amyloid peptides (Aβ(1-37/44), in the intracellular and extracellular space, that alters proteostasis and triggers Aβ modification (e.g., by reactive oxygen species (ROS)) into toxic intermediate, misfolded soluble Aβ peptides, Aβ dimers and Aβ oligomers. The toxic intermediate Aβ products aggregate into progressively less toxic and less soluble protofibrils, fibrils and senile plaques. This review focuses on peptides that inhibit toxic Aβ oligomerization, Aβ aggregation into fibrils, or stabilize Aβ peptides in non-toxic oligomers, and discusses their potential for AD treatment.

Metabolic Abnormalities of Erythrocytes as a Risk Factor for Alzheimer's Disease.

Alzheimer's disease (AD) is a slowly progressive, neurodegenerative disorder of uncertain etiology. According to the amyloid cascade hypothesis, accumulation of non-soluble amyloid β peptides (Aβ) in the Central Nervous System (CNS) is the primary cause initiating a pathogenic cascade leading to the complex multilayered pathology and clinical manifestation of the disease. It is, therefore, not surprising that the search for mechanisms underlying cognitive changes observed in AD has focused exclusively on the brain and Aβ-inducing synaptic and dendritic loss, oxidative stress, and neuronal death. However, since Aβ depositions were found in normal non-demented elderly people and in many other pathological conditions, the amyloid cascade hypothesis was modified to claim that intraneuronal accumulation of soluble Aβ oligomers, rather than monomer or insoluble amyloid fibrils, is the first step of a fatal cascade in AD. Since a characteristic reduction of cerebral perfusion and energy metabolism occurs in patients with AD it is suggested that capillary distortions commonly found in AD brain elicit hemodynamic changes that alter the delivery and transport of essential nutrients, particularly glucose and oxygen to neuronal and glial cells. Another important factor in tissue oxygenation is the ability of erythrocytes (red blood cells, RBC) to transport and deliver oxygen to tissues, which are first of all dependent on the RBC antioxidant and energy metabolism, which finally regulates the oxygen affinity of hemoglobin. In the present review, we consider the possibility that metabolic and antioxidant defense alterations in the circulating erythrocyte population can influence oxygen delivery to the brain, and that these changes might be a primary mechanism triggering the glucose metabolism disturbance resulting in neurobiological changes observed in the AD brain, possibly related to impaired cognitive function. We also discuss the possibility of using erythrocyte biochemical aberrations as potential tools that will help identify a risk factor for AD.

Role of presynaptic calcium stores for neural network dysfunction in Alzheimer's disease.

Role of presynaptic calcium stores for neural network dysfunction in Alzheimer's disease.

Preparation of Stable Amyloid-β Oligomers Without Perturbative Methods.

Soluble amyloid-β (Aβ) oligomers have become a focal point in the study of Alzheimer's disease due to their ability to elicit cytotoxicity. A number of recent studies have concentrated on the structural characterization of soluble Aβ oligomers to gain insight into their mechanism of toxicity. Consequently, providing reproducible protocols for the preparation of such oligomers is of utmost importance. The method presented in this chapter details a protocol for preparing an Aβ oligomer, with a primarily disordered secondary structure, without the need for chemical modification or amino acid substitution. Due to the stability of these disordered Aβ oligomers and the reproducibility with which they form, they are amenable for biophysical and high-resolution structural characterization.