Soluble Aβo Research Articles

Soluble Abeta produces an increase in cytosolic and mitochondrial calcium in astrocytes in vivo, in part by direct effect in astroglial cells

AbstractBackgroundBy using in vivo multiphoton microscopy, we have previously reported that astrocytic cytosolic resting calcium (Ca2+) is globally elevated in a transgenic mouse model of cerebral β‐amyloidosis (APPswe/PS1dE9) (PMID:19251629). However, this phenomenon is independent of the proximity of astrocytes to amyloid β (Aβ) plaques. In addition, soluble Aβ oligomers (Aβo) are thought to mediate neuronal toxicity by increasing neuronal calcium and causing synapse loss. For those reasons, we hypothesized that Aβo, rather than Aβ plaques, contribute to the Ca2+ insult in astroglial cytosol and mitochondria in vivo.MethodTo test this hypothesis, we topically applied naturally secreted soluble Aβo (conditioned medium from cultured transgenic primary neurons) onto the brain surface of 4‐6‐month old wild‐type mice. We investigated changes on astrocyte and mitochondrial Ca2+ levels relative to baseline via intravital multiphoton imaging, by using a ratiometric genetically‐encoded FRET‐based Ca2+ indicator (Yellow Cameleon 3.6) targeted to astroglial cytosol or mitochondria.ResultWe observed that Ca2+ levels dramatically increased upon topical soluble Aβo application, in all cytosolic astrocyte compartments (soma, branches and end feet), but also in astrocyte mitochondria. Aβ‐immunodepleted transgenic conditioned media and wild‐type conditioned media did not alter astrocyte cytosolic or mitochondrial Ca2+, supporting the specificity of the observed effects. Furthermore, we have also seen this effect in cultured astrocytes, suggesting that Aβo is directly interacting with astroglial cells, and not through an effect on other cell types, such as neurons or microglia. In addition, we previously reported that upon blocking the mitochondrial calcium uniporter (MCU) with Ru360, calcium levels in neuronal mitochondria were restored (PMID:32358564). However, there was still an increase of mitochondrial calcium in astrocytes in presence of soluble Aβo and Ru360 in vivo. Thus, soluble Aβo is disrupting calcium levels in astrocytes through a different mechanism.ConclusionUltimately, these results together support a detrimental role of Aβo which leads to astrocyte Ca2+ dyshomeostasis in vivo, implying that Aβo are involved in the astrocytic dysfunction observed in AD. Future studies will continue to explore the source of soluble Aβo‐induced Ca2+ increase, including intracellular Ca2+ stores and channels involved, which could have therapeutic value.

Hyperspectral analysis of amyloid beta (Aβ) evolutional changes in preclinical to late‐stage Alzheimer’s disease using matched brain and retinal tissue

AbstractBackgroundAlzheimer’s disease (AD) is a progressive, degenerative brain disorder that leads to cognitive impairment and eventually death. It is marked by buildup of abnormal amyloid aggregates (Aβ) in the brain. This study aimed to elucidate the neuropathologic sequence and corresponding [RetiSpec] hyperspectral signals of Aβ evolutional changes in preclinical to late‐stage AD using retinal and brain samples.MethodWe used brain‐ and retina‐matched human tissue samples from the NIH Brain Bank at Johns Hopkins. Study groups included: No AD neuropathologic change with negative AD neuropathology (n = 15); Low‐level AD neuropathologic change with positive AD neuropathology (n = 15); High level AD neuropathologic change with positive AD neuropathology (n = 15). To validate retinal hyperspectral imaging (rHSI) detection of Aβ in these retinal samples, we quantified Aβ oligomers (AβOs) in human retinal tissue. Immunohistochemistry was performed. We used humanized, affinity‐matured, IgG2 mAβ selective biotinylated antibody (ACU‐193) to detect soluble AβOs in retinal and brain tissues followed by objective quantification of ACU193 retinal staining by performing annotations and data extraction using QuPath software. One‐way ANOVA and t‐tests were performed to compare between controls and AD stages.ResultWhen assessing ACU193 staining present from retinal tissue samples, we observed 29‐53% retinal tissue area positivity in controls and 15‐72% retinal tissue area positivity in the AD group. Level of AD pathology demonstrated an effect on the rHSI spectra (450‐600nm) in regions near the optic disc and the ventral periphery: intermediate AD pathology showed the strongest rHSI signature (decrease in optical transmittance) followed by high, then low AD pathology samples. Correlation of the Braak neurofibrillary tangle stage and ΔOD offered a Pearson r score of 0.79 (p = 0.004).ConclusionThis study was the first to assess and quantify AβOs in clinically, neuropathologically, and hyperspectrally characterized postmortem matched brain and retinal tissues. Progression of well‐established neuropathologically classified AD stages (e.g Braak) may correlate with retinal AβO levels measured using rHSI. Certain retina regions that share nervous tissue with the brain (optic disc, periphery) are likely more sensitive to rHSI‐mediated detection of AD pathology. Larger samples are needed to comprehensively assess and quantify retinal AβOs.

Hippocampal injections of soluble amyloid-beta oligomers alter electroencephalographic activity during wake and slow-wave sleep in rats

BackgroundSoluble amyloid-beta oligomers (Aβo) begin to accumulate in the human brain one to two decades before a clinical diagnosis of Alzheimer’s disease (AD). The literature supports that soluble Aβo are implicated in synapse and neuronal losses in the brain regions such as the hippocampus. This region importantly contributes to explicit memory, the first type of memory affected in AD. During AD preclinical and prodromal stages, people are also experiencing wake/sleep alterations such as insomnia (e.g., difficulty initiating sleep, decreased sleep duration), excessive daytime sleepiness, and sleep schedule modifications. In addition, changes in electroencephalographic (EEG) activity during wake and sleep have been reported in AD patients and animal models. However, the specific contribution of Aβo to wake/sleep alterations is poorly understood and was investigated in the present study.MethodsChronic hippocampal injections of soluble Aβo were conducted in male rats and combined with EEG recording to determine the progressive impact of Aβ pathology specifically on wake/sleep architecture and EEG activity. Bilateral injections were conducted for 6 consecutive days, and EEG acquisition was done before, during, and after Aβo injections. Immunohistochemistry was used to assess neuron numbers in the hippocampal dentate gyrus (DG).ResultsAβo injections did not affect the time spent in wakefulness, slow wave sleep (SWS), and paradoxical sleep but altered EEG activity during wake and SWS. More precisely, Aβo increased slow-wave activity (SWA; 0.5–5 Hz) and low-beta activity (16–20 Hz) during wake and decreased theta (5–9 Hz) and alpha (9–12 Hz) activities during SWS. Moreover, the theta activity/SWA ratio during wake and SWS was decreased by Aβo. These effects were significant only after 6 days of Aβo injections and were found with alterations in neuron counts in the DG.ConclusionsWe found multiple modifications of the wake and SWS EEG following Aβo delivery to the hippocampus. These findings expose a specific EEG signature of Aβ pathology and can serve the development of non-invasive and cost-effective markers for the early diagnosis of AD or other amyloid-related diseases.

Using giant plasma membrane vesicles (GMPVs) to study age-dependent changes in amyloid-β oligomer toxicity.

Aging is a major risk factor associated with several human ailments, including some forms of cancer, autoimmunity, and neurodegeneration. Longitudinal changes in the multicellular organisms, including issues with genomic stability and protein oxidation, have been extensively reported. However, age-dependent changes in plasma membrane lipids and how they contribute to age-related phenotypes are less understood. Moreover, mechanistic understanding about how age-dependent lipidic changes can seed or exacerbate the toxicity of aggregating protein, such as amyloid-β (Aβ) in Alzheimer's disease, is of particular interest. Indeed, certain isolated detergents, fatty acids, and lipids have been shown to nucleate soluble Aβ oligomers (Aβos) with notable cytotoxicity, but little has been done to understand how changes in complex mixtures such as plasma membrane lipids scaffold toxic oligomers in a longitudinal manner. To deconvolute how cellular age can influence the cytotoxic properties of Aβos, giant-plasma membrane derived vesicles (GPMVs) will be collected from cultured cells at various passage numbers and at senescence. Those GMPVs will then be used as lipidic scaffolds for soluble Aβos and the Aβo-GPMV complexes will be treated to cultured cells to monitor their responses. Specifically, Aβo-GPMV toxicity will be monitored by observing changes in cellular respiration, senescence, and/or cell death. The driving hypothesis is that GPMVs derived from aged cells (at high-passage numbers) will seed enhanced toxicity of Aβ oligomers compared to the young (low/medium passage number) counterparts.

New Alzheimer models for drug screening based on improved human amyloid beta (1-42) oligomer preparations.

The etiology of Alzheimer's disease (AD) is poorly understood. A growing body of literature suggests that amyloid beta oligomers (AβO) as the root cause of this disease. Here, we describe new translational in vitro and in vivo models of AD, induced by minute amount of in-house preparation of human AβO. AβO were reproducibly prepared from human Aβ 1-42 monomers. Rodent primary neurons were used to assess the neurotoxic activities of AβO in vitro and elderly wild-type mice administered by a single intracerebral injection AβO in vivo. The oligomeric preparations were characterized by SDS-page, electron microscopy and dot-plot tests. In vitro, AβO induced a dose-dependent neurodegeneration on rodent primary neurons based on various read-outs. Interestingly, neurotoxicity was greater with AβO than fibrillar Aβ, while Aβ monomers did not induce any neuronal damage. AβO-induced neurotoxicity was significantly attenuated by brain-derived neurotrophic factor (BDNF). In vivo, a single intracerebral microinjection of AβO in 18-month-old wild-type mice, led to a significant memory impairment, synaptic loss in hippocampus and increased secretion of brain proinflammatory cytokines 15 days post administration. Moreover, the memory deficits were significantly reversed with Donepezil used as reference drug. In conclusion, we characterized new tools for drug screening based on the soluble AβO hypothesis of AD. In vivo, this new non-inherited Alzheimer model can be used both to evaluate disease modifying and symptomatic drugs. Finally, these models are also valuable tools to understand the mechanisms underlying AD.

Synthetic amyloid-β oligomers drive early pathological progression of Alzheimer’s disease in nonhuman primates

SummaryAs an insidious and slowly progressive neurodegenerative disorder, Alzheimer’s disease (AD) uniquely develops in humans but fails in other species. Therefore, it has been challenged to rebuild human AD in animals, including in non-human primates. Here, we bilaterally delivered synthetic Aβ oligomers (AβOs) into the cerebral parenchyma of cynomolgus monkeys, which rapidly drove the formation of massive Aβ plaques and concomitant neurofibrillary tangles in the cynomolgus brain. The amyloid and tau pathology as well as their co-occurrence in AβO-monkeys were reminiscent of those in patients with AD. In addition, the activated astrocytes and microglia surrounding Aβ plaques indicated the triggered neuroinflammation. The degenerative neurons and synapses around Aβ plaques also emerged in cynomolgus brain. Together, soluble AβOs caused the cascade of pathologic events associated with AD in monkeys as occurred in patients at the early phase, which could facilitate the development of a promising animal model for human AD in non-human primates.

Hyperactivity Induced by Soluble Amyloid-β Oligomers in the Early Stages of Alzheimer's Disease.

Soluble amyloid-beta oligomers (Aβo) start to accumulate in the human brain one to two decades before any clinical symptoms of Alzheimer's disease (AD) and are implicated in synapse loss, one of the best predictors of memory decline that characterize the illness. Cognitive impairment in AD was traditionally thought to result from a reduction in synaptic activity which ultimately induces neurodegeneration. More recent evidence indicates that in the early stages of AD synaptic failure is, at least partly, induced by neuronal hyperactivity rather than hypoactivity. Here, we review the growing body of evidence supporting the implication of soluble Aβo on the induction of neuronal hyperactivity in AD animal models, in vitro, and in humans. We then discuss the impact of Aβo-induced hyperactivity on memory performance, cell death, epileptiform activity, gamma oscillations, and slow wave activity. We provide an overview of the cellular and molecular mechanisms that are emerging to explain how Aβo induce neuronal hyperactivity. We conclude by providing an outlook on the impact of hyperactivity for the development of disease-modifying interventions at the onset of AD.

Amyloid-β oligomers in cellular models of Alzheimer's disease.

Amyloid-β (Aβ) dysmetabolism is tightly associated with pathological processes in Alzheimer's disease (AD). Currently, it is thought that, in addition to Aβ fibrils that give rise to plaque formation, Aβ aggregates into non-fibrillar soluble oligomers (AβOs). Soluble AβOs have been extensively studied for their synaptotoxic and neurotoxic properties. In this review, we discuss physicochemical properties of AβOs and their impact on different brain cell types in AD. Additionally, we summarize three decades of studies with AβOs, providing a compelling bulk of evidence regarding cell-specific mechanisms of toxicity. Cellular models may lead us to a deeper understanding of the detrimental effects of AβOs in neurons and glial cells, putatively shedding light on the development of innovative therapies for AD.

Systematic and standardized comparison of reported amyloid-β receptors for sufficiency, affinity, and Alzheimer's disease relevance

Oligomeric assemblies of amyloid-β (Aβ) peptide (Aβo) in the brains of individuals with Alzheimer's disease (AD) are toxic to neuronal synapses. More than a dozen Aβ receptor candidates have been suggested to be responsible for various aspects of the molecular pathology and memory impairment in mouse models of AD. A lack of consistent experimental design among previous studies of different receptor candidates limits evaluation of the relative roles of these candidates, producing some controversy within the field. Here, using cell-based assays with several Aβ species, including Aβo from AD brains obtained by autopsy, we directly compared the Aβ-binding capacity of multiple receptor candidates while accounting for variation in expression and confirming cell surface expression. In a survey of 15 reported Aβ receptors, only cellular prion protein (PrPC), Nogo receptor 1 (NgR1), and leukocyte immunoglobulin-like receptor subfamily B member 2 (LilrB2) exhibited direct binding to synaptotoxic assemblies of synthetic Aβ. Both PrPC and NgR1 preferentially bound synaptotoxic oligomers rather than nontoxic monomers, and the method of oligomer preparation did not significantly alter our binding results. Hippocampal neurons lacking both NgR1 and LilrB2 exhibited a partial reduction of Aβo binding, but this reduction was lower than in neurons lacking PrPC under the same conditions. Finally, binding studies with soluble Aβo from human AD brains revealed a strong affinity for PrPC, weak affinity for NgR1, and no detectable affinity for LilrB2. These findings clarify the relative contributions of previously reported Aβ receptors under controlled conditions and highlight the prominence of PrPC as an Aβ-binding site.

Targeting Soluble Amyloid Oligomers in Alzheimer's Disease through Disordered Prion Peptides

Cognitive decline in Alzheimer's Disease (AD) and many other age-related dementias have long been associated with the presence of insoluble amyloid plaques that disrupt normal synaptic functioning. However, recent studies have revealed that synapse impairment from AD is much more potently associated with soluble amyloid-β oligomers (aβo), rather than from insoluble fibrils. Soluble oligomers are able to adopt heterogeneous structures that are irregular and promiscuously bind to membrane proteins, thereby dysregulating downstream amyloid assemblies such as tau. Multiple screening methods have identified cellular prion protein (PrPc) as a putative target of aβo, and subsequent studies have confirmed a pathophysiological pathway in AD involving aβo-PrPc binding. However, aβ monomers and insoluble fibrils do not bind to PrPc, thus the disordered binding domain of PrPc can be used to isolate aβo assemblies. In order to exploit these interactions, we designed biomimetic PrPc peptides that complex soluble aβo in replica-exchange molecular dynamics simulations. While peptides are unlikely to act as AD therapeutics, they can help inform the types of side-chain interactions that drive soluble amyloid formation for use in next-generation therapeutics that cross the blood-brain barrier. Similarly, targeting aβo through disordered PrPc peptides can enable the tracking of soluble oligomers through fluorescent tagging during aβ fibril formation. To the extent possible, results are compared to solid-state NMR spectroscopy of aβo-PrPc complexes, where heterogeneous structures are identified. Taken together, this study highlights how small disordered peptides modulate pathological protein behaviors in AD and other degenerative diseases where soluble amyloid oligomers, rather than insoluble fibrils, drive cell toxicity.

Doxycycline counteracts neuroinflammation restoring memory in Alzheimer's disease mouse models.

β-Amyloid oligomers (AβOs) and neuroinflammation are 2 main culprits to counteract in Alzheimer's disease (AD). Doxycycline (DOXY) is a second generation antibiotic of the tetracycline class that are promising drugs tested in many clinical trials for a number of different pathologies. DOXY is endowed with antiamyloidogenic properties and better crosses the blood-brain barrier, but its efficacy has never been tested in AD mice. We herein show that 15- to 16-month-old APP/PS1dE9 (APP/PS1) AD mice receiving DOXY under different treatment regimens recovered their memory without plaque reduction. An acute DOXY treatment was, also, sufficient to improve APP/PS1 mouse memory, suggesting an action against soluble AβOs. This was confirmed in an AβO-induced mouse model, where the AβO-mediated memory impairment was abolished by a DOXY pretreatment. Although AβOs induce memory impairment through glial activation, assessing the anti-inflammatory action of DOXY, we found that in both the AβO-treated and APP/PS1 mice, the memory recovery was associated with a lower neuroinflammation. Our data promote DOXY as a hopeful repositioned drug counteracting crucial neuropathological AD targets.

Stabilized Low-n Amyloid-β Oligomers Induce Robust Novel Object Recognition Deficits Associated with Inflammatory, Synaptic, and GABAergic Dysfunction in the Rat

With current treatments for Alzheimer's disease (AD) only providing temporary symptomatic benefits, disease modifying drugs are urgently required. This approach relies on improved understanding of the early pathophysiology of AD. A new hypothesis has emerged, in which early memory loss is considered a synapse failure caused by soluble amyloid-β oligomers (Aβo). These small soluble Aβo, which precede the formation of larger fibrillar assemblies, may be the main cause of early AD pathologies. The aim of the current study was to investigate the effect of acute administration of stabilized low-n amyloid-β1-42 oligomers (Aβo1-42) on cognitive, inflammatory, synaptic, and neuronal markers in the rat. Female and male Lister Hooded rats received acute intracerebroventricular (ICV) administration of either vehicle or 5 nmol of Aβo1-42 (10μL). Cognition was assessed in the novel object recognition (NOR) paradigm at different time points. Levels of inflammatory (IL-1β, IL-6, TNF-α), synaptic (PSD-95, SNAP-25), and neuronal (n-acetylaspartate, parvalbumin-positive cells) markers were investigated in different brain regions (prefrontal and frontal cortex, striatum, dorsal and ventral hippocampus). Acute ICV administration of Aβo1-42 induced robust and enduring NOR deficits. These deficits were reversed by acute administration of donepezil and rolipram but not risperidone. Postmortem analysis revealed an increase in inflammatory markers, a decrease in synaptic markers and parvalbumin containing interneurons in the frontal cortex, with no evidence of widespread neuronal loss. Taken together the results suggest that acute administration of soluble low-n Aβo may be a useful model to study the early mechanisms involved in AD and provide us with a platform for testing novel therapeutic approaches that target the early underlying synaptic pathology.

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.

TRPA1 channels promote astrocytic Ca2+ hyperactivity and synaptic dysfunction mediated by oligomeric forms of amyloid-\u03b2 peptide

BackgroundExcessive synaptic loss is thought to be one of the earliest events in Alzheimer’s disease (AD). However, the key mechanisms that maintain plasticity of synapses during adulthood or initiate synapse dysfunction in AD remain unknown. Recent studies suggest that astrocytes contribute to functional changes observed during synaptic plasticity and play a major role in synaptic dysfunction but astrocytes behavior and involvement in early phases of AD remained largely undefined.MethodsWe measure astrocytic calcium activity in mouse CA1 hippocampus stratum radiatum in both the global astrocytic population and at a single cell level, focusing in the highly compartmentalized astrocytic arbor. Concurrently, we measure excitatory post-synaptic currents in nearby pyramidal neurons.ResultsWe find that application of soluble Aβ oligomers (Aβo) induced fast and widespread calcium hyperactivity in the astrocytic population and in the microdomains of the astrocyte arbor. We show that astrocyte hyperactivity is independent of neuronal activity and is repaired by transient receptor potential A1 (TRPA1) channels blockade. In return, this TRPA1 channels-dependent hyperactivity influences neighboring CA1 neurons triggering an increase in glutamatergic spontaneous activity. Interestingly, in an AD mouse model (APP/PS1–21 mouse), astrocyte calcium hyperactivity equally takes place at the beginning of Aβ production, depends on TRPA1 channels and is linked to CA1 neurons hyperactivity.ConclusionsOur experiments demonstrate that astrocytes contribute to early Aβo toxicity exhibiting a global and local Ca2+ hyperactivity that involves TRPA1 channels and is related to neuronal hyperactivity. Together, our data suggest that astrocyte is a frontline target of Aβo and highlight a novel mechanism for the understanding of early synaptic dysregulation induced by soluble Aβo species.

TARGETING OF TOXIC AMYLOID-BETA OLIGOMER SPECIES BY MONOCLONAL ANTIBODY PMN310: PRECISION DRUG DESIGN FOR ALZHEIMER’S DISEASE

Current evidence suggests that progressive neurodegeneration in Alzheimer's disease (AD) may be catalyzed by the prion-like propagation of soluble toxic amyloid-beta oligomers (AβO) rather than plaque burden. Binding of Aβ monomers and/or fibrils by therapeutic antibodies has been associated with suboptimal efficacy and adverse events (e.g. ARIA-E) in clinical trials. These observations suggest that antibodies capable of specifically or selectively neutralizing toxic AβO are needed to achieve improved efficacy and safety. Computational algorithms were developed to identify epitope sequences and conformations likely to be exposed in toxic AβO but not on monomers or fibrils. The binding profile of monoclonal antibodies raised against predicted epitopes was assessed by surface plasmon resonance (SPR) analysis and immunohistochemistry (IHC). Inhibition of oligomer propagation was assessed by thioflavin-T fluorescence in vitro. Neutralization of AβO neurotoxicity by the antibodies was evaluated in cultures of primary neurons in vitro and in wild-type mice injected intracerebroventricularly with AβO in vivo. Screening of IgG clones raised against 5 distinct AβO epitopes yielded antibodies with the desired profile of selective binding to synthetic oligomers vs monomers, preferential binding to native soluble AβO in CSF and brain extracts of AD patients compared to controls by SPR, and lack of plaque reactivity by IHC on unfixed AD brain sections. Activity assays led to the identification of PMN310 as a lead antibody capable of blocking the propagation and neurotoxicity of AβO in vitro and protecting mice against the AβO-induced loss of short term memory formation in vivo. Computational modeling allowed for the identification and generation of monoclonal antibodies against AβO-specific epitopes. Antibody PMN310 was selected as a lead candidate on the basis of its ability to selectively target and neutralize AβO with no significant cross-reactivity to monomers or fibrils. These unique characteristics distinguish PMN310 from Aβ-reactive antibodies currently undergoing clinical trials, and are designed to address observed issues of efficacy and safety.

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.

Visual and fluorescent assays for selective detection of beta-amyloid oligomers based on the inner filter effect of gold nanoparticles on the fluorescence of CdTe quantum dots

Beta-amyloid (Aβ) peptides are the major constituents of senile plaques in the brains of Alzheimer’s disease (AD) patients. Aβ monomers (AβMs) can coalesce to form small, soluble oligomers (AβOs), followed by reorganization and assembly into long, thread-like fibrils (AβFs). Recently, soluble AβOs have been regarded as reliable molecular biomarkers for the diagnosis of AD because of their high toxicity for neuronal synapse and high concentration levels in the brains of AD patients. In this work, we reported a label-free, sensitive and selective method for visual and fluorescent detection of AβOs based on the inner filter effect (IFE) of gold nanoparticles (AuNPs) on the fluorescence of CdTe quantum dots (QDs). Specifically, the fluorescence of CdTe QDs was quenched significantly by AuNPs through the IFE. PrP(95–110), an AβOs-specific binding peptide from cellular prion protein, triggered the aggregation and color change of AuNPs suspension; thus, the IFE of AuNPs on the fluorescence of CdTe QDs was weakened and the fluorescence intensity was recovered. However, in the presence of AβOs, the specific interaction of AβOs and PrP(95–110) prevented the absorption of PrP(95–110) onto the surface of AuNPs. As a result, the aggregation of AuNPs was inhibited and the fluorescence intensity of CdTe QDs was quenched again. This label-free method is specific for detection of AβOs but not for AβMs and AβFs. The detection limits were found to be 0.5nM for the visual assay and 0.2nM for the fluorescent detection. We believe that this work would be valuable for many investigations related to AD diagnosis and drug discovery.

Extracellular and intraneuronal HMW-AbetaOs represent a molecular basis of memory loss in Alzheimer's disease model mouse

BackgroundSeveral lines of evidence indicate that memory loss represents a synaptic failure caused by soluble amyloid β (Aβ) oligomers. However, the pathological relevance of Aβ oligomers (AβOs) as the trigger of synaptic or neuronal degeneration, and the possible mechanism underlying the neurotoxic action of endogenous AβOs remain to be determined.ResultsTo specifically target toxic AβOs in vivo, monoclonal antibodies (1A9 and 2C3) specific to them were generated using a novel design method. 1A9 and 2C3 specifically recognize soluble AβOs larger than 35-mers and pentamers on Blue native polyacrylamide gel electrophoresis, respectively. Biophysical and structural analysis by atomic force microscopy (AFM) revealed that neurotoxic 1A9 and 2C3 oligomeric conformers displayed non-fibrilar, relatively spherical structure. Of note, such AβOs were taken up by neuroblastoma (SH-SY5Y) cell, resulted in neuronal death. In humans, immunohistochemical analysis employing 1A9 or 2C3 revealed that 1A9 and 2C3 stain intraneuronal granules accumulated in the perikaryon of pyramidal neurons and some diffuse plaques. Fluoro Jade-B binding assay also revealed 1A9- or 2C3-stained neurons, indicating their impending degeneration. In a long-term low-dose prophylactic trial using active 1A9 or 2C3 antibody, we found that passive immunization protected a mouse model of Alzheimer's disease (AD) from memory deficits, synaptic degeneration, promotion of intraneuronal AβOs, and neuronal degeneration. Because the primary antitoxic action of 1A9 and 2C3 occurs outside neurons, our results suggest that extracellular AβOs initiate the AD toxic process and intraneuronal AβOs may worsen neuronal degeneration and memory loss.ConclusionNow, we have evidence that HMW-AβOs are among the earliest manifestation of the AD toxic process in mice and humans. We are certain that our studies move us closer to our goal of finding a therapeutic target and/or confirming the relevance of our therapeutic strategy.

Amyloid-β Protein Oligomer at Low Nanomolar Concentrations Activates Microglia and Induces Microglial Neurotoxicity

Amyloid-β Protein Oligomer at Low Nanomolar Concentrations Activates Microglia and Induces Microglial Neurotoxicity