Conformation-dependent single-chain variable fragment antibodies specifically recognize beta-amyloid oligomers

Increasing evidence indicates that beta-amyloid (Aβ) oligomers rather than monomers or fibrils are the major toxic agents that specifically inhibit synaptic plasticity and long-term potentiation (LTP) in Alzheimer’s disease (AD). Neutralization of Aβ oligomeric toxicity was found to reverse memory deficits. Here, we report four single-chain variable fragment (scFv) antibodies isolated from the naive human scFv library by phage display that specifically recognized Aβ oligomers but not monomers and fibrils. These conformation-dependent scFv antibodies inhibit both Aβ fibrillation and cytotoxicity and bind to the same type of eptitope displayed on the Aβ oligomers. Such scFv antibodies specifically targeting toxic Aβ oligomers may have potential therapeutic and diagnostic applications for AD.     

Isolation and characterization of antibody fragments selective for specific protein morphologies from nanogram antigen samples

We developed atomic force microscope (AFM)‐based protocols that enable isolation and characterization of antibody‐based reagents that selectively bind target protein variants using low nanogram amounts or less of unpurified starting material. We isolated single‐chain antibody fragments (scFvs) that specifically recognize an oligomeric beta‐amyloid (Aβ) species correlated with Alzheimer's disease (AD) using only a few nanograms of an enriched but not purified sample obtained from human AD brain tissue. We used several subtractive panning steps to remove all phage binding nondesired antigens and then used a single positive panning step using minimal antigen. We also used AFM to characterize the specificity of the isolated clones, again using minimal material, selecting the C6 scFv based on expression levels. We show that C6 selectively binds cell and brain‐derived oligomeric Aβ. The protocols described are readily adapted to isolating antibody‐based reagents against other antigenic targets with limited availability. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 463–471, 2013     

Identification of low molecular weight pyroglutamate A{beta} oligomers in Alzheimer disease: a novel tool for therapy and diagnosis

N-terminally truncated Aβ peptides starting with pyroglutamate (AβpE3) represent a major fraction of all Aβ peptides in the brain of Alzheimer disease (AD) patients. AβpE3 has a higher aggregation propensity and stability and shows increased toxicity compared with full-length Aβ. In the present work, we generated a novel monoclonal antibody (9D5) that selectively recognizes oligomeric assemblies of AβpE3 and studied the potential involvement of oligomeric AβpE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost nondetectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal and blood vessel staining was observed. Using a novel sandwich ELISA significantly decreased levels of oligomers in plasma samples from patients with AD compared with healthy controls were identified. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall Aβ plaque load and AβpE3 levels, and normalized behavioral deficits. These data indicate that 9D5 is a therapeutically and diagnostically effective monoclonal antibody targeting low molecular weight AβpE3 oligomers.     

Extracellular phosphorylation of the amyloid β-peptide promotes formation of toxic aggregates during the pathogenesis of Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia and associated with progressive deposition of amyloid β-peptides (Aβ) in the brain. Aβ derives by sequential proteolytic processing of the amyloid precursor protein by β- and γ-secretases. Rare mutations that lead to amino-acid substitutions within or close to the Aβ domain promote the formation of neurotoxic Aβ assemblies and can cause early-onset AD. However, mechanisms that increase the aggregation of wild-type Aβ and cause the much more common sporadic forms of AD are largely unknown. Here, we show that extracellular Aβ undergoes phosphorylation by protein kinases at the cell surface and in cerebrospinal fluid of the human brain. Phosphorylation of serine residue 8 promotes formation of oligomeric Aβ assemblies that represent nuclei for fibrillization. Phosphorylated Aβ was detected in the brains of transgenic mice and human AD brains and showed increased toxicity in Drosophila models as compared with non-phosphorylated Aβ. Phosphorylation of Aβ could represent an important molecular mechanism in the pathogenesis of the most common sporadic form of AD.     

Orally administrated cinnamon extract reduces β-amyloid oligomerization and corrects cognitive impairment in Alzheimer's disease animal models

An increasing body of evidence indicates that accumulation of soluble oligomeric assemblies of β-amyloid polypeptide (Aβ) play a key role in Alzheimer's disease (AD) pathology. Specifically, 56 kDa oligomeric species were shown to be correlated with impaired cognitive function in AD model mice. Several reports have documented the inhibition of Aβ plaque formation by compounds from natural sources. Yet, evidence for the ability of common edible elements to modulate Aβ oligomerization remains an unmet challenge. Here we identify a natural substance, based on cinnamon extract (CEppt), which markedly inhibits the formation of toxic Aβ oligomers and prevents the toxicity of Aβ on neuronal PC12 cells. When administered to an AD fly model, CEppt rectified their reduced longevity, fully recovered their locomotion defects and totally abolished tetrameric species of Aβ in their brain. Furthermore, oral administration of CEppt to an aggressive AD transgenic mice model led to marked decrease in 56 kDa Aβ oligomers, reduction of plaques and improvement in cognitive behavior. Our results present a novel prophylactic approach for inhibition of toxic oligomeric Aβ species formation in AD through the utilization of a compound that is currently in use in human diet.     

Amelioration of amyloid load by anti-Abeta single-chain antibody in Alzheimer mouse model

Parenteral immunization of transgenic mouse models of Alzheimer disease (AD) with synthetic amyloid beta-peptide (Abeta) prevented or reduced Abeta deposits and attenuated their memory and learning deficits. A clinical trial of immunization with synthetic Abeta, however, was halted due to brain inflammation, presumably induced by a toxic Abeta, T-cell- and/or Fc-mediated immune response. Another issue relating to such immunizations is that some AD patients may not be able to raise an adequate immune response to Abeta vaccination due to immunological tolerance or age-associated decline. Because peripheral administration of antibodies against Abeta also induced clearance of amyloid plaques in the model mice, injection of humanized Abeta antibodies has been proposed as a possible therapy for AD. By screening a human single-chain antibody (scFv) library for Abeta immunoreactivity, we have isolated a scFv that specifically reacts with oligomeric Abeta as well as amyloid plaques in the brain. The scFv inhibited Abeta amyloid fibril formation and Abeta-mediated cytotoxicity in vitro. We have tested the efficacy of the human scFv in a mouse model of AD (Tg2576 mice). Relative to control mice, injections of the scFv into the brain of Tg2576 mice reduced Abeta deposits. Because scFvs lack the Fc portion of the immunoglobulin molecule, human scFvs against Abeta may be useful to treat AD patients without eliciting brain inflammation.     

Alzheimer's disease: synapses gone cold

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by insidious cognitive decline and memory dysfunction. Synapse loss is the best pathological correlate of cognitive decline in AD and mounting evidence suggests that AD is primarily a disease of synaptic dysfunction. Soluble oligomeric forms of amyloid beta (Aβ), the peptide that aggregates to form senile plaques in the brain of AD patients, have been shown to be toxic to neuronal synapses both in vitro and in vivo. Aβ oligomers inhibit long-term potentiation (LTP) and facilitate long-term depression (LTD), electrophysiological correlates of memory formation. Furthermore, oligomeric Aβ has also been shown to induce synapse loss and cognitive impairment in animals. The molecular underpinnings of these observations are now being elucidated, and may provide clear therapeutic targets for effectively treating the disease. Here, we review recent findings concerning AD pathogenesis with a particular focus on how Aβ impacts synapses.     

Expression in drosophila of tandem amyloid β peptides provides insights into links between aggregation and neurotoxicity

The generation and subsequent aggregation of amyloid β (Aβ) peptides play a crucial initiating role in the pathogenesis of Alzheimer disease (AD). The two main isoforms of these peptides have 40 (Aβ(40)) or 42 residues (Aβ(42)), the latter having a higher propensity to aggregate in vitro and being the main component of the plaques observed in vivo in AD patients. We have designed a series of tandem dimeric constructs of these Aβ peptides to probe the manner in which changes in the aggregation kinetics of Aβ affect its deposition and toxicity in a Drosophila melanogaster model system. The levels of insoluble aggregates were found to be substantially elevated in flies expressing the tandem constructs of both Aβ(40) and Aβ(42) compared with the equivalent monomeric peptides, consistent with the higher effective concentration, and hence increased aggregation rate, of the peptides in the tandem repeat. A unique feature of the Aβ(42) constructs, however, is the appearance of high levels of soluble oligomeric aggregates and a corresponding dramatic increase in their in vivo toxicity. The toxic nature of the Aβ(42) peptide in vivo can therefore be attributed to the higher kinetic stability of the oligomeric intermediate states that it populates relative to those of Aβ(40) rather than simply to its higher rate of aggregation.     

Antifibrillizing agents catalyze the formation of unstable intermediate aggregates of beta‐amyloid

Although Alzheimer's disease (AD) is characterized by the extracellular deposition of fibrillar aggregates of beta‐amyloid (Aβ), transient oligomeric species of Aβ are increasingly implicated in the pathogenesis of AD. Natively unfolded monomeric Aβ can misfold and progressively assemble into fibrillar aggregates, following a well‐established “on pathway” seeded‐nucleation mechanism. Here, we show that three simple saccharides, mannose, sucrose, and raffinose, alter Aβ aggregation kinetics and morphology. The saccharides inhibit formation of Aβ fibrils but promote formation of various oligomeric aggregate species through different “off pathway” aggregation mechanisms at 37°C but not at 60°C. The various oligomeric Aβ aggregates formed when coincubated with the different saccharides are morphologically distinct but all are toxic toward SH‐SY5Y human neuroblastoma cells, increasing the level of toxicity and greatly prolonging toxicity compared with Aβ alone. As a wide variety of anti‐Aβ aggregation strategies are being actively pursued as potential therapeutics for AD, these studies suggest that care must be taken to ensure that the therapeutic agents also block toxic oligomeric Aβ assembly as well as inhibit fibril formation. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Human apolipoprotein A–I binds amyloid-β and prevents Aβ-induced neurotoxicity

Aggregates of the amyloid-β peptide (Aβ) play a central role in the pathogenesis of Alzheimer's disease (AD). Identification of proteins that physiologically bind Aβ and modulate its aggregation and neurotoxicity could lead to the development of novel disease-modifying approaches in AD. By screening a phage display peptide library for high affinity ligands of aggregated Aβ_1–42, we isolated a peptide homologous to a highly conserved amino acid sequence present in the N-terminus of apolipoprotein A–I (apoA-I). We show that purified human apoA-I and Aβ form non-covalent complexes and that interaction with apoA-I affects the morphology of amyloid aggregates formed by Aβ. Significantly, Aβ/apoA-I complexes were also detected in cerebrospinal fluid from AD patients. Interestingly, apoA-I and apoA-I-containing reconstituted high density lipoprotein particles protect hippocampal neuronal cultures from Aβ-induced oxidative stress and neurodegeneration. These results suggest that human apoA-I modulates Aβ aggregation and Aβ-induced neuronal damage and that the Aβ-binding domain in apoA-I may constitute a novel framework for the design of inhibitors of Aβ toxicity.     

Biflavonoids are superior to monoflavonoids in inhibiting amyloid-β toxicity and fibrillogenesis via accumulation of nontoxic oligomer-like structures

Polymerization of monomeric amyloid-β peptides (Aβ) into soluble oligomers and insoluble fibrils is one of the major pathways triggering the pathogenesis of Alzheimer's disease (AD). Using small molecules to prevent the polymerization of Aβ peptides can, therefore, be an effective therapeutic strategy for AD. In this study, we investigate the effects of mono- and biflavonoids in Aβ42-induced toxicity and fibrillogenesis and find that the biflavonoid taiwaniaflavone (TF) effectively and specifically inhibits Aβ toxicity and fibrillogenesis. Compared to TF, the monoflavonoid apigenin (AP) is less effective and less specific. Our data show that differential effects of the mono- and biflavonoids in Aβ fibrillogenesis correlate with their varying cytoprotective efficacies. We also find that other biflavonoids, namely, 2',8'-biapigenin, amentoflavone, and sumaflavone, can also effectively inhibit Aβ toxicity and fibrillogenesis, implying that the participation of two monoflavonoids in a single biflavonoid molecule enhances their activity. Biflavonoids, while strongly inhibiting Aβ fibrillogenesis, accumulate nontoxic Aβ oligomeric structures, suggesting that these are off-pathway oligomers. Moreover, TF abrogates the toxicity of preformed Aβ oligomers and fibrils, indicating that TF and other biflavonoids may also reduce the toxicity of toxic Aβ species. Altogether, our data clearly show that biflavonoids, possibly because of the possession of two Aβ binders separated by an appropriate size linker, are likely to be promising therapeutics for suppressing Aβ toxicity.     

Isolation of a human single chain antibody fragment against oligomeric alpha-synuclein that inhibits aggregation and prevents alpha-synuclein-induced toxicity

Protein misfolding and aggregation are pathological aspects of numerous neurodegenerative diseases. Aggregates of alpha-synuclein are major components of the Lewy bodies and Lewy neurites associated with Parkinson's Disease (PD). A natively unfolded protein, alpha-synuclein can adopt different aggregated morphologies, including oligomers, protofibrils and fibrils. The small oligomeric aggregates have been shown to be particularly toxic. Antibodies that neutralize the neurotoxic aggregates without interfering with beneficial functions of monomeric alpha-synuclein can be useful therapeutics. We were able to isolate single chain antibody fragments (scFvs) from a phage displayed antibody library against the target antigen morphology using a novel biopanning technique that utilizes atomic force microscopy (AFM) to image and immobilize specific morphologies of alpha-synuclein. The scFv described here binds only to an oligomeric form of alpha-synuclein and inhibits both aggregation and toxicity of alpha-synuclein in vitro. This scFv can have potential therapeutic value in controlling misfolding and aggregation of alpha-synuclein in vivo when expressed intracellularly in dopaminergic neurons as an intrabody.     

Anti-oligomeric single chain variable domain antibody differentially affects huntingtin and alpha-synuclein aggregates

Huntington's and Parkinson's diseases are both neurodegenerative disorders caused at least in part by misfolding and aggregation of huntingtin (htt) and alpha-synuclein, respectively. Here we use a single chain antibody fragment (scFv) isolated against oligomeric alpha-synuclein to probe similarities and differences between the aggregation and toxic mechanisms of htt and alpha-synuclein. When incubated with htt, the scFv both blocks formation of and promotes dissociation of fibrillar aggregates, but stabilizes formation of cytotoxic oligomeric aggregates. Previous studies with monomeric alpha-synuclein showed the scFv prevented fibrillar aggregation, but blocked toxicity of oligomeric aggregates. These divergent effects suggest the toxic mechanisms of oligomeric aggregates differ among amyloidogenic protein species.     

Chronic intranasal treatment with an anti-Aβ(30-42) scFv antibody ameliorates amyloid pathology in a transgenic mouse model of Alzheimer's disease

Amyloid-beta peptide (Aβ)-directed active and passive immunization therapeutic strategies reduce brain levels of Aβ, decrease the severity of beta-amyloid plaque pathology and reverse cognitive deficits in mouse models of Alzheimer's disease (AD). As an alternative approach to passive immunization with full IgG molecules, single-chain variable fragment (scFv) antibodies can modulate or neutralize Aβ-related neurotoxicity and inhibit its aggregation in vitro. In this study, we characterized a scFv derived from a full IgG antibody raised against the C-terminus of Aβ, and studied its passage into the brains of APP transgenic mice, as well as its potential to reduce Aβ-related pathology. We found that the scFv entered the brain after intranasal application, and that it bound to beta-amyloid plaques in the cortex and hippocampus of APP transgenic mice. Moreover, the scFv inhibited Aβ fibril formation and Aβ-mediated neurotoxicity in vitro. In a preventative therapeutic approach chronic intranasal treatment with scFv reduced congophilic amyloid angiopathy (CAA) and beta-amyloid plaque numbers in the cortex of APPswe/PS1dE9 mice. This reduction of CAA and plaque pathology was associated with a redistribution of brain Aβ from the insoluble fraction to the soluble peptide pool. Due to their lack of the effector domain of full IgG, scFv may represent an alternative tool for the treatment of Aβ-related pathology without triggering Fc-mediated effector functions. Additionally, our observations support the possibility that Aβ-directed immunotherapy can reduce Aβ deposition in brain vessels in transgenic mice.     

Intraneuronal Aβ as a trigger for neuron loss: can this be translated into human pathology?

In the present review, we summarize the current achievements of modelling early intraneuronal Aβ (amyloid β-peptide) accumulation in transgenic mice with the resulting pathological consequences. Of special importance will be to discuss recent developments and the translation of the results to AD (Alzheimer's disease). N-terminally truncated AβpE3 (Aβ starting with pyroglutamate at position 3) represents a major fraction of all Aβ peptides in the brain of AD patients. Recently, we generated a novel mAb (monoclonal antibody), 9D5, that selectively recognizes oligomeric assemblies of AβpE3 and demonstrated the potential involvement of oligomeric AβpE3 in vivo using transgenic mouse models as well as human brains from sporadic and familial AD cases. 9D5 showed an unusual staining pattern with almost non-detectable plaques in sporadic AD patients and non-demented controls. Interestingly, in sporadic and familial AD cases prominent intraneuronal staining was observed. Moreover, passive immunization of 5XFAD mice with 9D5 significantly reduced overall Aβ levels and stabilized behavioural deficits. In summary, we have demonstrated that intraneuronal Aβ is a valid risk factor in model systems and AD patients. This feature of AD pathology was successful in identifying novel low-molecular-mass oligomeric Aβ-specific antibodies for diagnosis and therapy.     

Intrahippocampal administration of a domain antibody that binds aggregated amyloid-β reverses cognitive deficits produced by diet-induced obesity

BackgroundThe prevalence of high fat diets (HFD), diet-induced obesity (DIO) and Type 2 diabetes continues to increase, associated with cognitive impairment in both humans and rodent models. Mechanisms transducing these impairments remain largely unknown: one possibility is that a common mechanism may be involved in the cognitive impairment seen in obese and/or diabetic states and in dementia, specifically Alzheimer's disease (AD). DIO is well established as a risk factor for development of AD. Oligomeric amyloid-β (Aβ) is neurotoxic, and we showed that intrahippocampal oligomeric Aβ produces cognitive and metabolic dysfunction similar to that seen in DIO or diabetes. Moreover, animal models of DIO show elevated brain Aβ, a hallmark of AD, suggesting that this may be one source of cognitive impairment in both conditions.MethodsIntrahippocampal administration of a novel anti-Aβ domain antibody for aggregated Aβ, or a control domain antibody, to control or HFD-induced DIO rats. Spatial learning measured in a conditioned contextual fear (CCF) task after domain antibody treatment; postmortem, hippocampal NMDAR and AMPAR were measured.ResultsDIO caused impairment in CCF, and this impairment was eliminated by intrahippocampal administration of the active domain antibody. Measurement of hippocampal proteins suggests that DIO causes dysregulation of hippocampal AMPA receptors, which is also reversed by acute domain antibody administration.ConclusionsOur findings support the concept that oligomeric Aβ within the hippocampus of DIO animals may not only be a risk factor for development of AD but may also cause cognitive impairment before the development of dementia.General Significance and InterestOur work integrates the engineering of domain antibodies with conformational- and sequence-specificity for oligomeric amyloid beta with a clinically relevant model of diet-induced obesity in order to demonstrate not only the pervasive effects of obesity on several aspects of brain biochemistry and behavior, but also the bioengineering of a successful treatment against the long-term detrimental effects of a pre-diabetic state on the brain. We show for the first time that cognitive impairment linked to obesity and/or insulin resistance may be due to early accumulation of oligomeric beta-amyloid in the brain, and hence may represent a pre-Alzheimer's state.     

Fibrillar seeds alleviate amyloid-β cytotoxicity by omitting formation of higher-molecular-weight oligomers

Amyloid-β (Aβ) peptides can exist in distinct forms including monomers, oligomers and fibrils, consisting of increased numbers of monomeric units. Among these, Aβ oligomers are implicated as the primary toxic species as pointed by multiple lines of evidence. It has been suggested that toxicity could be rendered by the soluble higher-molecular-weight (high-n) Aβ oligomers. Yet, the most culpable form in the pathogenesis of Alzheimer’s disease (AD) remains elusive. Moreover, the potential interaction among the insoluble fibrils that have been excluded from the responsible aggregates in AD development, Aβ monomers and high-n oligomers is undetermined. Here, we report that insoluble Aβ fibrillar seeds can interact with Aβ monomers at the stoichiometry of 1:2 (namely, each Aβ molecule of seed can bind to two Aβ monomers at a time) facilitating the fibrillization by omitting the otherwise mandatory formation of the toxic high-n oligomers during the fibril maturation. As a result, the addition of exogenous Aβ fibrillar seeds is seen to rescue neuronal cells from Aβ cytotoxicity presumably exerted by high-n oligomers, suggesting an unexpected protective role of Aβ fibrillar seeds.     

Crocetin inhibits beta-amyloid fibrillization and stabilizes beta-amyloid oligomers

Aggregation of a peptide, beta-amyloid (Aβ), is a hallmark molecular process found in Alzheimer’s disease (AD). During Aβ aggregation, oligomeric and fibrillar Aβ are formed, and these molecular self-assembly steps are implicated in generation of toxic effects in AD. Crocetin is a natural carotenoid dicarboxyl acid displaying various pharmaceutical effects and may be co-localized with Aβ mediated by human serum albumin. In the study presented here, we examined the effects of crocetin on Aβ aggregation in three different molecular pathways. Our results demonstrate that crocetin inhibited Aβ fibril formation and destabilized pre-formed Aβ fibrils. Moreover, crocetin caused stabilization of Aβ oligomers and prevented their conversion into Aβ fibrils. Our study reveals potential pathological and pharmaceutical implication of crocetin in AD and suggests possible application of crocetin for currently limited structural studies on unstable Aβ oligomers.     

Direct in Vivo Intracellular Selection of Conformation-sensitive Antibody Domains Targeting Alzheimer's Amyloid-β Oligomers

The development of conformation-sensitive antibody domains targeting the misfolding beta amyloid (Aβ) peptide is of great interest for research into Alzheimer's disease (AD).We describe the direct selection, by the Intracellular Antibody Capture Technology (IACT), of a panel of anti-Aβ single chain Fv antibody fragments (scFvs), targeting pathologically relevant conformations of Aβ. A LexA-Aβ1-42 fusion protein was expressed in yeast cells, as the “intracellular antigen”. Two different scFv antibody libraries (Single Pot Libraries of Intracellular Antibodies, SPLINT) were used for the intracellular selections: (i) a naïve library, derived from a natural, non-immune, source of mouse antibody variable region (V) genes; and (ii) an immune library constructed from the repertoire of antibody V genes of Aβ-immunized mice. This led to the isolation of 18 different anti-Aβ scFvs, which bind Aβ both in the yeast cell, as well as in vitro, if used as purified recombinant proteins. Surprisingly, all the anti-Aβ scFvs isolated are conformation-sensitive, showing a high degree of specificity towards Aβ oligomers with respect to monomeric Aβ, while also displaying some degree of sequence-specificity, recognizing either the N-terminal or the C-terminal part of Aβ1-42; in particular, the scFvs selected from Aβ-immune SPLINT library show a relevant N-terminal epitope bias. Representative candidates from this panel of the anti-Aβ scFvs were shown to recognize in vivo-produced Aβ “deposits” in histological sections from human AD brains and to display good neutralization properties, significantly inhibiting Aβ oligomer-induced toxicity and synaptic binding of Aβ oligomers in neuronal cultured cells. The properties of these anti-Aβ antibody domains, as well as their direct availability for intra- or extra-cellular “genetic delivery” make them ideally suited for new experimental approaches to study and image the intracellular processing and trafficking of Aβ oligomers.     

Calpain cleavage and inactivation of the sodium calcium exchanger‐3 occur downstream of Aβ in Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by pathological deposits of β‐amyloid (Aβ) in senile plaques, intracellular neurofibrillary tangles (NFTs) comprising hyperphosphorylated aggregated tau, synaptic dysfunction and neuronal death. Substantial evidence indicates that disrupted neuronal calcium homeostasis is an early event in AD that could mediate synaptic dysfunction and neuronal toxicity. Sodium calcium exchangers (NCXs) play important roles in regulating intracellular calcium, and accumulating data suggests that reduced NCX function, following aberrant proteolytic cleavage of these exchangers, may contribute to neurodegeneration. Here, we show that elevated calpain, but not caspase‐3, activity is a prominent feature of AD brain. In addition, we observe increased calpain‐mediated cleavage of NCX3, but not a related family member NCX1, in AD brain relative to unaffected tissue and that from other neurodegenerative conditions. Moreover, the extent of NCX3 proteolysis correlated significantly with amounts of Aβ1–42. We also show that exposure of primary cortical neurons to oligomeric Aβ1–42 results in calpain‐dependent cleavage of NCX3, and we demonstrate that loss of NCX3 function is associated with Aβ toxicity. Our findings suggest that Aβ mediates calpain cleavage of NCX3 in AD brain and therefore that reduced NCX3 activity could contribute to the sustained increases in intraneuronal calcium concentrations that are associated with synaptic and neuronal dysfunction in AD.