Soluble Amyloid-beta Oligomers Research Articles

Prestimulation of Microglia Through TLR4 Pathway Promotes Interferon Beta Expression in a Rat Model of Alzheimer's Disease.

Soluble amyloid beta (Aβ) oligomers are the most common forms of Aβ in the early stage of Alzheimer's disease (AD). They are highly toxic to the neurons but their capability to activate microglia remains controversial. Microglia develop two distinct phenotypes, classic (M1) and alternative (M2). Tuning of microglia to the alternative (anti-inflammatory) state is of major interest in treatment of neuroinflammatory disease. This study aimed to assess tuning the microglia to produce interferon beta (IFN-β) as an anti-inflammatory cytokine through TLR4 pathway in a rat model of AD. Microglial BV-2 cells were treated with 1μg/ml lipopolysaccharides (LPS), Monophosphoryl lipid A (MPL), or vehicles for 24h, and then incubated with Aβ oligomer. After 24h, cell pellets were harvested and TIR-domain-containing adapter-inducing interferon-β (TRIF), interferon regulatory factor 3 (IRF3), and IFN-β levels were measured. The ligands/vehicle were microinjected into the right ventricle of male Wistar rats every 3days. Two weeks later, an osmotic pump filled with oligomeric Aβ/vehicle was implanted in the left ventricle. After 2weeks, TRIF, IRF3, and IFN-β levels were measured in the hippocampal tissue. TNF-α and IFN-β levels were assessed in the hippocampus using immunohistochemistry. The oligomeric Aβ did not change TRIF, IRF3, and IFN-β levels in both cell culture and hippocampal tissue. However, pretreatment with LPS or MPL increased the level of these proteins. BV-2 cells morphologically express M1 state in presence of higher dose of Aβ oligomer (10μM). Pretreatment with LPS or MPL decreased the TNF-α and increased the number of IFN-β positive cells in the hippocampus of Aβ-treated rats. In conclusion, pretreatment with low dose TLR4 agonists could induce microglia to produce neuroprotective cytokines including IFN-β which may be considered as a potential strategy to combat neuronal degeneration in AD.

A hydrogel biosensor for high selective and sensitive detection of amyloid-beta oligomers.

BackgroundAlzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive cognitive and memory impairment. It is the most common neurological disease that causes dementia. Soluble amyloid-beta oligomers (AβO) in blood or cerebrospinal fluid (CSF) are the pathogenic biomarker correlated with AD.MethodsA simple electrochemical biosensor using graphene oxide/gold nanoparticles (GNPs) hydrogel electrode was developed in this study. Thiolated cellular prion protein (PrPC) peptide probe was immobilized on GNPs of the hydrogel electrode to construct an AβO biosensor. Electrochemical impedance spectroscopy was utilized for AβO analysis.ResultsThe specific binding between AβO and PrPC probes on the hydrogel electrode resulted in an increase in the electron-transfer resistance. The biosensor showed high specificity and sensitivity for AβO detection. It could selectively differentiate AβO from amyloid-beta (Aβ) monomers or fibrils. Meanwhile, it was highly sensitive to detect as low as 0.1 pM AβO in artificial CSF or blood plasma. The linear range for AβO detection is from 0.1 pM to 10 nM.ConclusionThis biosensor could be used as a cost-effective tool for early diagnosis of AD due to its high electrochemical performance and bionic structure.

Early changes in hyppocampal neurogenesis induced by soluble Ab1-42 oligomers

Alzheimer's disease is characterized by the loss of neurons, the accumulation of intracellular neurofibrillary tangles and extracellular amyloid plaques in the brain. However, there are contradicting data on differences in neurogenesis at the onset of the disease or before the formation of amyloid plaques. As awareness of the importance of the pre-symptom phase in neurodegenerative diseases grows in the context of early diagnosis and pathogenesis, we analyzed the critical periods of adult hippocampal neurogenesis at an early stage under the action of soluble Ab1-42 beta-amyloid. The proliferation, migration and neuronal cells survival were evaluated in mice with an injection of soluble amyloid beta-oligomers. It was found that the injection of Ab1-42 oligomers causes a decrease in cell proliferation in the mouse hippocampus. Despite the preservation of the neuroblast pool in animals after beta-amyloid injection, the process of radial migration is disrupted, and an increase in apoptosis in the neurogenic niche was revealed. Thus, our results demonstrate damage of neurogenesis critical stages: the progenitor cells, neuroblast migration, the integration of immature neurons, and the survival of neurons under application of soluble beta-amyloid oligomers. The obtained data indicate decline in proliferation rate in the subgranular zone, that is accompanied by ectopic differentiation and disturbed migration, producing, apparently, abnormal neurons that have lower survival rates. That could lead to a decrease in mature neurons numbers and the number of cells in the granular layer of the dentate gyrus.

Amyloid β oligomers (AβOs) in Alzheimer’s disease

The causative role of amyloid β 1-42 (Aβ42) aggregation in the pathogenesis of Alzheimer's disease (AD) has been under debate for over 25years. Primarily, scientific efforts have focused on the dyshomeostasis between production and clearance of Aβ42. This imbalance may result from mutations either in genes for the substrate, i.e., amyloid precursor protein or in genes encoding presenilin, the enzyme of the reaction that generates Aβ42. Currently, it is supposed that soluble oligomers of amyloid beta (AβOs) and not fibrillar Aβ42 within neuritic plaques may be the toxic factors acting on a very early stage of AD, perhaps even initiating pathological cascade. For example, soluble AβOs isolated from AD patients' brains reduced number of synapses, inhibited long-term potentiation, and enhanced long-term synaptic depression in brain regions responsible for memory in animal models of AD. Concentrations of AβOs in the cerebrospinal fluid (CSF) of AD patients are often reported higher than in non-demented controls, and show a negative correlation with mini-mental state examination scores. Furthermore, increased Aβ42/oligomer ratio in the CSF of AD/MCI patients indicated that the presence of soluble AβOs in CSF may be linked to lowering of natively measured monomeric Aβ42 by epitopes masking, and hence, concentrations of AβOs in the CSF are postulated to as useful AD biomarkers.

CROSSBETA STABLE Aβ OLIGOMERS: DISEASE-RELEVANT DRUG TARGETS AND VERSATILE REFERENCE STANDARDS IN BIOMARKER ASSAYS IN AD

There is accumulating evidence that soluble amyloid beta (Aβ) oligomers, rather than the amyloid fibrils, are the principal pathogenic species in Alzheimer disease (AD). Because they exhibit acute synaptic toxicity and induce neurodegenerative processes, and because the Aβ oligomer levels in cerebrospinal fluid appear to correlate with disease severity and progression, these species constitute an optimal drug target and possible useful diagnostic marker in early AD. Unfortunately, the instability and limited characterization of oligomer preparations have seriously impeded research and development in this field. Crossbeta has addressed this problem by developing a technology to reproducibly generate stable soluble Aß1–42 oligomers that are devoid of monomeric or fibrillar Aß and show biophysical properties indistinguishable from the native species. Furthermore, Crossbeta oligomers demonstrate typical functionality of pathological Aβ species such as neurotoxicity, in vivo LTP depression and induction of inflammatory response. The availability of the stable oligomers opens previously inaccessible avenues for R&D. For example, it enabled high throughput screening of drug-like compound libraries. Derived from a 100k compound screening effort, small molecule CBB68 was identified and shown to neutralize oligomer-induced neurotoxicity in rat primary neurons and to rescue the synaptic deficit induced by Aβ oligomers in vivo as well as in vitro. In particular, the in vivo LTP assay demonstrated the efficacy and brain penetration of CBB68 after intravenous administration. In another application, Crossbeta's oligomers have also been used to select shark antibodies that specifically recognize a conformational oligomeric epitope and bind to Aß1–42 oligomers with high affinity (sub-nM), but not to the monomers or fibrils. This antibody provides potential to selectively quantify Aß1–42 oligomers in patient material as part of a high sensitivity biomarker assay together with the stable Crossbeta oligomers as calibrator. Furthermore, the oligomers are excellent research tools, e.g. in disease-relevant bioassays. We show and conclude that the stable oligomers enable multiple applications and constitute optimum drug targets.

In silico investigation on the inhibition of Aβ42 aggregation by Aβ40 peptide by potential of mean force study

Recent experimental data revealed that small, soluble Amyloid beta (Aβ42) oligomers, especially dimers impair synaptic plasticity and memory leading to Alzheimer’s disease. Here, we have studied dimerization of Aβ42/Aβ42 homo-dimer and Aβ40/Aβ42 hetero-dimer in terms of free energy profile by all-atom simulations using the ff99SB force field. We have found that in the presence of Aβ40 peptide, there exists a strong tendency to form a hetero-dimer with Aβ42 peptide, suggesting that a possible co-oligomerization. Furthermore, we have investigated the effects of Aβ40 on the Aβ42 peptide. Our study also shows that in presence of Aβ40, the beta-content of Aβ42 monomer is reduced. Additionally, certain residues important for bending in Aβ42 peptide attained an increased flexibility in the presence of Aβ40. The salt-bridge destabilization also manifested the impact of Aβ40 on Aβ42 peptide as a whole. Based on this, one may expect that Aβ40 inhibits the aggregation propensity of Aβ42. Moreover, the binding free energy obtained by the molecular mechanics–Poisson–Boltzmann surface area method also revealed a strong affinity between the two isoforms thereby suggests that Aβ40 binding induces conformational change in Aβ42. Our results suggest that co-oligomerization of Aβ isoforms may play a substantial role in Alzheimer’s disease.

Overexpression of EphB2 in hippocampus rescues impaired NMDA receptors trafficking and cognitive dysfunction in Alzheimer model

Alzheimer’s disease (AD) is a progressive neurodegenerative disease, which affects more and more people. But there is still no effective treatment for preventing or reversing the progression of the disease. Soluble amyloid-beta (Aβ) oligomers, also known as Aβ-derived diffusible ligands (ADDLs) play an important role in AD. Synaptic activity and cognition critically depend on the function of glutamate receptors. Targeting N-methyl-D-aspartic acid (NMDA) receptors trafficking and its regulation is a new strategy for AD early treatment. EphB2 is a key regulator of synaptic localization of NMDA receptors. Aβ oligomers could bind to the fibronectin repeats domain of EphB2 and trigger EphB2 degradation in the proteasome. Here we identified that overexpression of EphB2 with lentiviral vectors in dorsal hippocampus improved impaired memory deficits and anxiety or depression-like behaviors in APPswe/PS1-dE9 (APP/PS1) transgenic mice. Phosphorylation and surface expression of GluN2B-containing NMDA receptors were also improved. Overexpression of EphB2 also rescued the ADDLs-induced depletion of the expression of EphB2 and GluN2B-containing NMDA receptors trafficking in cultured hippocampal neurons. These results suggest that improving the decreased expression of EphB2 and subsequent GluN2B-containing NMDA receptors trafficking in hippocampus may be a promising strategy for AD treatment.

Management of Alzheimers Disease: Role of Existing Therapies, Traditional Medicines and New Treatment Targets

Dementia, a common problem in elderly, is rising worldwide including India. Alzheimer’s disease is the main etiology for dementia. Currently anticholinesterases, N-methyl-D-aspartate receptor antagonists, disease modifying agents form the mainstay of treatment of Alzheimer’s disease. However, recent breakthroughs in Alzheimer’s disease research have given multiple new treatment targets. This review summarizes the role of existing therapies and their limitations, role of traditional medicines and new treatment targets. The newer agents include molecules targeting tau protein like modulators of τ-kinases or phosphatases, kinase inhibitors, τ-aggregation inhibitors. It also includes molecules which targets amyloid plaques like, inhibitors or modulators of the secretases, amyloid-β aggregation inhibitors and amyloid-plaque degradation enhancers. Role of nootropics, statins and antipsychotics in the management of Alzheimer’s disease is limited and lacks conclusive evidence. Studies with vaccines have shown reduced amyloid beta and τ-pathology, neutralized soluble amyloid beta oligomers. But search for the appropriate vaccine for Alzheimer’s disease is still on. Antioxidants, metal chelators, Metal-protein attenuating compounds, metal complexes have also shown some potential. Traditional medicines like huperzine A and galantamine have been used for management of Alzheimer’s disease. Traditional medicinal system in India particularly Ayurveda mentions neuroprotective actions of plant drugs like Brahmi, Shankhapushpi, Amla, Guduchi, Tulsi, Ashwagandha and Haritaki, which can help in improving the treatment armamentarium for Alzheimer’s disease.The understanding of Alzheimer’s disease neurobiology is improving now and the search for better treatment options is still on. This provides an opportunity for more research on integrated approach using existing therapies, traditional and alternative medicines with newer treatments and potential candidates for management of Alzheimer’s disease.

Cerebral dopamine neurotrophic factor alleviates Aβ25-35-induced endoplasmic reticulum stress and early synaptotoxicity in rat hippocampal cells

Alzheimer’s disease (AD) is an age-related progressive neurodegenerative disease, and early stage AD is characterized by synaptic dysfunction generally ascribed to soluble oligomers of amyloid-beta (Aβ). Neurotrophic factors are promising for AD treatment and are integrally involved in neuronal growth, survival and maintenance. Cerebral dopamine neurotrophic factor (CDNF) was recently discovered to have beneficial effects on long-term memory. The present study explored the synaptoprotective effects of CDNF in Aβ-treated primary hippocampal cells. Immunofluorescent analysis of synaptophysin and postsynaptic density protein 95 (PSD95) puncta densities in the group of pretreatment with CDNF before Aβ exposure revealed significant improvements compared to Aβ group. In addition, pretreatment with CDNF reduced the expression levels of endoplasmic reticulum (ER) stress-related proteins, including Bip (also known as GRP78), phosphorylation of eukaryotic translation initiation factor 2 subunit α (peIF2α), phosphorylation of c-Jun N-terminal kinase (pJNK), and cleaved caspase 3, which are increased by Aβ treatment at early stage. Our results revealed protective effects of CDNF on Aβ-induced synaptotoxicity and ER stress, implying that CDNF may protect against Aβ-induced synaptotoxicity through suppression of ER stress. CDNF could be a potential drug candidate for early AD treatment.

Astrocytic GluN2A and GluN2B Oppose the Synaptotoxic Effects of Amyloid-β1-40 in Hippocampal Cells.

Early-stage Alzheimer's disease (AD) is characterized by synaptic dysfunction, a phenomenon in which soluble oligomers of amyloid-beta (Aβ) and N-methyl-D-aspartate receptor (NMDAR) are implicated. Here, we demonstrated that astrocytes express NMDARs and therefore have the potential to modulate the synaptotoxic actions of Aβ. We found that specific pharmacological antagonism of two of the major NMDAR subunits, GluN2A and GluN2B, exacerbates Aβ-induced synaptotoxicity suggesting, for the first time, that astrocytic GluN2A and GluN2B mediate synaptoprotection. From the perspective of the pathogenic mechanisms of Alzheimer's disease, in which Aβ and NMDAR play significant roles, these observations are striking since neuronal GluN2A and GluN2B are well known modulators of neurodegeneration. We did initial studies to understand the basis for the differential effects of astrocytic and neuronal GluN2A and GluN2B in the promotion of synapse survival, and identified a neurotrophin produced by astrocytes, nerve growth factor β (β-NGF), as a likely mediator of the synaptoprotective effects of astrocytic GluN2A and GluN2B activation. The results presented suggest that astrocytes may be suitable druggable targets for the prevention and/or delay of the synaptic loss that occurs during early stages of AD.

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps.

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer's disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (Aβo) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble Aβo correlate with memory deficits in AD models and humans. The Aβo-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated Aβo infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning Aβo-induced memory deficits at the onset of AD. Since soluble Aβo aggregate relatively fast into insoluble Aβ fibrils that correlate poorly with the clinical state of patients, soluble Aβo are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of Aβo and continuous infusion of Aβo antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of Aβo in pre-dementia patients.

Neurodegeneration in an Animal Model of Chronic Amyloid-beta Oligomer Infusion Is Counteracted by Antibody Treatment Infused with Osmotic Pumps

Decline in hippocampal-dependent explicit memory (memory for facts and events) is one of the earliest clinical symptom of Alzheimer's disease (AD). It is well established that synapse loss and ensuing neurodegeneration are the best predictors for memory impairments in AD. Latest studies have emphasized the neurotoxic role of soluble amyloid-beta oligomers (Aβo) that begin to accumulate in the human brain approximately 10 to 15 yr before the clinical symptoms become apparent. Many reports indicate that soluble Aβo correlate with memory deficits in AD models and humans. The Aβo-induced neurodegeneration observed in neuronal and brain slice cultures has been more challenging to reproduce in many animal models. The model of repeated Aβo infusions shown here overcome this issue and allow addressing two key domains for developing new disease modifying therapies: identify biological markers to diagnose early AD, and determine the molecular mechanisms underpinning Aβo-induced memory deficits at the onset of AD. Since soluble Aβo aggregate relatively fast into insoluble Aβ fibrils that correlate poorly with the clinical state of patients, soluble Aβo are prepared freshly and injected once per day during six days to produce marked cell death in the hippocampus. We used cannula specially design for simultaneous infusions of Aβo and continuous infusion of Aβo antibody (6E10) in the hippocampus using osmotic pumps. This innovative in vivo method can now be used in preclinical studies to validate the efficiency of new AD therapies that might prevent the deposition and neurotoxicity of Aβo in pre-dementia patients.

Small molecule NPT-440-1 inhibits ionic flux through Aβ1 -42 pores: Implications for Alzheimer's disease therapeutics

Increased levels of soluble amyloid-beta (Aβ) oligomers are suspected to underlie Alzheimer's disease (AD) pathophysiology. These oligomers have been shown to form multi-subunit Aβ pores in bilayers and induce uncontrolled, neurotoxic, ion flux, particularly calcium ions, across cellular membranes that might underlie cognitive impairment in AD. Small molecule interventions that modulate pore activity could effectively prevent or ameliorate their toxic activity. Here we examined the efficacy of a small molecule, NPT-440-1, on modulating amyloid pore permeability. Co-incubation of B103 rat neuronal cells with NPT-440-1 and Aβ1-42 prevented calcium influx. In purified lipid bilayers, we show that a 10-15min preincubation, prior to membrane introduction, was required to prevent conductance. Thioflavin-T and circular dichroism both suggested a reduction in Aβ1-42 β-sheet content during this incubation period. Combined with previous studies on site-specific amino acid substitutions, these results suggest that pharmacological modulation of Aβ1-42 could prevent amyloid pore-mediated AD pathogenesis.

Soluble amyloid beta oligomers block the learning-induced increase in hippocampal sharp wave-ripple rate and impair spatial memory formation.

Post-learning hippocampal sharp wave-ripples (SWRs) generated during slow wave sleep are thought to play a crucial role in memory formation. While in Alzheimer’s disease, abnormal hippocampal oscillations have been reported, the functional contribution of SWRs to the typically observed spatial memory impairments remains unclear. These impairments have been related to degenerative synaptic changes produced by soluble amyloid beta oligomers (Aβos) which, surprisingly, seem to spare the SWR dynamics during routine behavior. To unravel a potential effect of Aβos on SWRs in cognitively-challenged animals, we submitted vehicle- and Aβo-injected mice to spatial recognition memory testing. While capable of forming short-term recognition memory, Aβ mice exhibited faster forgetting, suggesting successful encoding but an inability to adequately stabilize and/or retrieve previously acquired information. Without prior cognitive requirements, similar properties of SWRs were observed in both groups. In contrast, when cognitively challenged, the post-encoding and -recognition peaks in SWR occurrence observed in controls were abolished in Aβ mice, indicating impaired hippocampal processing of spatial information. These results point to a crucial involvement of SWRs in spatial memory formation and identify the Aβ-induced impairment in SWRs dynamics as a disruptive mechanism responsible for the spatial memory deficits associated with Alzheimer’s disease.

Europium as an inhibitor of Amyloid-β(1-42) induced membrane permeation

Soluble Amyloid-beta (Aβ) oligomers are a source of cytotoxicity in Alzheimer’s disease (AD). The toxicity of Aβ oligomers may arise from their ability to interact with and disrupt cellular membranes mediated by GM1 ganglioside receptors within these membranes. Therefore, inhibition of Aβ–membrane interactions could provide a means of preventing the toxicity associated with Aβ. Here, using Surface Plasmon field-enhanced Fluorescence Spectroscopy, we determine that the lanthanide, Europium III chloride (Eu3+), strongly binds to GM1 ganglioside-containing membranes and prevents the interaction with Aβ42 leading to a loss of the peptides ability to cause membrane permeation. Here we discuss the molecular mechanism by which Eu3+ inhibits Aβ42-membrane interactions and this may lead to protection of membrane integrity against Aβ42 induced toxicity.

AMYLOID ION CHANNEL TOXICITY: DISRUPTION OF B-SHEET STRUCTURE PREVENTS TOXIC IONIC FLUX THROUGH AB PORES

A common mechanism underlying Alzheimer's disease (AD) pathophysiology relies upon increased levels of soluble amyloid-beta (Aβ) oligomers. These oligomers induce uncontrolled ion flux across cellular membranes resulting in the dysregulation of ionic homeostasis, particularly calcium ions. High calcium concentration is known to cause neuronal degeneration and death that might underlie AD cognitive impairment. Previous 3D structural analysis, using atomic force microscopy (AFM), in our laboratory has shown that Aβ forms multi-subunit ion channels (Figure A, inset). These ion channel structures are complemented by the expected ionic permeability and single channel conductance of calcium and other cations in purified bilayer preparations. Amyloid ion channel structure-activity relationships are tightly interlinked; pharmacological and small molecule interventions that modulate channel structure could be then become an effective strategy to prevent their toxic activity. In this study, we examined pharmacologic intervention in reducing ionic flux through A β ion channels. Aβ(1-42) ion channels were reconstituted in planar lipid bilayers (PLB) and electrical conductance recordings were analyzed both in the presence as well as in the absence of a defined pharmacological agent. The pharmacological agent used in the study is a proprietary and experimental compound provided by Neuropore Therapies Inc. (La Jolla, CA). Short pre-incubation (∼10 min) of the compound with Aβ(1-42) effectively prevented ion channel conductance (Figure C). However, the compound added along with A β(1-42) showed no change in ionic conductance (Figure B) vs. the control with A β(1-42) alone (Figure A), even at much higher compound concentrations.

Targeting the proper amyloid-beta neuronal toxins: a path forward for Alzheimer's disease immunotherapeutics.

Levels of amyloid-beta monomer and deposited amyloid-beta in the Alzheimer’s disease brain are orders of magnitude greater than soluble amyloid-beta oligomer levels. Monomeric amyloid-beta has no known direct toxicity. Insoluble fibrillar amyloid-beta has been proposed to be an in vivo mechanism for removal of soluble amyloid-beta and exhibits relatively low toxicity. In contrast, soluble amyloid-beta oligomers are widely reported to be the most toxic amyloid-beta form, both causing acute synaptotoxicity and inducing neurodegenerative processes. None of the amyloid-beta immunotherapies currently in clinical development selectively target soluble amyloid-beta oligomers, and their lack of efficacy is not unexpected considering their selectivity for monomeric or fibrillar amyloid-beta (or both) rather than soluble amyloid-beta oligomers. Because they exhibit acute, memory-compromising synaptic toxicity and induce chronic neurodegenerative toxicity and because they exist at very low in vivo levels in the Alzheimer’s disease brain, soluble amyloid-beta oligomers constitute an optimal immunotherapeutic target that should be pursued more aggressively.

Experimental Traumatic Brain Injury Induces Rapid Aggregation and Oligomerization of Amyloid-Beta in an Alzheimer's Disease Mouse Model

Soluble amyloid-beta (Aβ) oligomers are hypothesized to be the pathogenic species in Alzheimer's disease (AD), and increased levels of oligomers in the brain subsequent to traumatic brain injury (TBI) may exacerbate secondary injury pathways and underlie increased risk of developing AD in later life. To determine whether TBI causes Aβ aggregation and oligomerization in the brain, we exposed triple transgenic AD model mice to controlled cortical impact injury and measured levels of soluble, insoluble, and oligomeric Aβ by enzyme-linked immunosorbent assay (ELISA) at 1, 3, and 7 days postinjury. TBI rapidly increased levels of both soluble and insoluble Aβ40 and Aβ42 in the injured cortex at 1 day postinjury. We confirmed previous findings that identified damaged axons as a major site of Aβ accumulation using both immunohistochemistry and biochemistry. We also report that soluble Aβ oligomers were significantly increased in the injured cortex, as demonstrated by both ELISA and Western blot. Interestingly, the mouse brain is able to rapidly clear trauma-induced Aβ, with both soluble and insoluble Aβ species returning to sham levels by 7 days postinjury. In conclusion, we demonstrate that TBI causes acute accumulation and aggregation of Aβ in the brain, including the formation of low- and high-molecular-weight Aβ oligomers. The formation and aggregation of Aβ into toxic species acutely after injury may play a role in secondary injury cascades after trauma and, chronically, may contribute to increased risk of developing AD in later life.

Sub-lethal levels of amyloid β-peptide oligomers decrease non-transferrin-bound iron uptake and do not potentiate iron toxicity in primary hippocampal neurons

Two major lesions are pathological hallmarks in Alzheimer's disease (AD): the presence of neurofibrillary tangles formed by intracellular aggregates of the hyperphosphorylated form of the cytoskeletal tau protein, and of senile plaques composed of extracellular aggregates of amyloid beta (Aβ) peptide. Current hypotheses regard soluble amyloid beta oligomers (AβOs) as pathological causative agents in AD. These aggregates cause significant calcium deregulation and mediate neurotoxicity by disrupting synaptic activity. Additionally, the presence of high concentrations of metal ions such as copper, zinc, aluminum and iron in neurofibrillary tangles and senile plaques, plus the fact that they accelerate the rate of formation of Aβ fibrils and AβOs in vitro, suggests that accumulation of these metals in the brain is relevant to AD pathology. A common cellular response to AβOs and transition metals such as copper and iron is the generation of oxidative stress, with the ensuing damage to cellular components. Using hippocampal neurons in primary culture, we report here the effects of treatment with AβOs on the (+)IRE and (-)IRE mRNA levels of the divalent metal transporter DMT1. We found that non-lethal AβOs concentrations decreased DMT1 (-)IRE without affecting DMT1 (+)IRE mRNA levels, and inhibited non-transferrin bound iron uptake. In addition, since both iron and AβOs induce oxidative damage, we studied whether their neurotoxic effects are synergistic. In the range of concentrations and times used in this study, AβOs did not potentiate iron-induced cell death while iron chelation did not decrease AβOs-induced cell death. The lack of synergism between iron and AβOs suggests that these two neurotoxic agents converge in a common target, which initiates signaling processes that promote neurodegeneration.

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.