Globular amyloid beta-peptide oligomer - a homogenous and stable neuropathological protein in Alzheimer's disease

Amyloid beta-peptide (Abeta)(1-42) oligomers have recently been discussed as intermediate toxic species in Alzheimer's disease (AD) pathology. Here we describe a new and highly stable Abeta(1-42) oligomer species which can easily be prepared in vitro and is present in the brains of patients with AD and Abeta(1-42)-overproducing transgenic mice. Physicochemical characterization reveals a pure, highly water-soluble globular 60-kDa oligomer which we named 'Abeta(1-42) globulomer'. Our data indicate that Abeta(1-42) globulomer is a persistent structural entity formed independently of the fibrillar aggregation pathway. It is a potent antigen in mice and rabbits eliciting generation of Abeta(1-42) globulomer-specific antibodies that do not cross-react with amyloid precursor protein, Abeta(1-40) and Abeta(1-42) monomers and Abeta fibrils. Abeta(1-42) globulomer binds specifically to dendritic processes of neurons but not glia in hippocampal cell cultures and completely blocks long-term potentiation in rat hippocampal slices. Our data suggest that Abeta(1-42) globulomer represents a basic pathogenic structural principle also present to a minor extent in previously described oligomer preparations and that its formation is an early pathological event in AD. Selective neutralization of the Abeta globulomer structure epitope is expected to have a high potential for treatment of AD.     

Immunotherapeutic strategies for prevention and treatment of Alzheimer's disease.

Pathologic examination in Alzheimer's disease (AD) shows a significant correlation between beta-amyloid peptide (AbetaP) deposition and the clinical severity of dementia. Formation of beta-amyloid (Abeta) is a complex kinetic and thermodynamic process, dependent on peptide-peptide interactions that may be modulated by other proteins. We found that site-directed antibodies toward peptide EFRH sequences 3-6 of the N-terminal region of AbetaP suppress in vitro formation of Abeta and dissolve already-formed fibrillar amyloid. These so-called chaperone-like properties of monoclonal antibodies led to the development of a new immunologic approach to AD treatment. The immunization procedure, based on phages displaying the EFRH epitope as antigen, induced anti-AbetaP antibodies that recognized the whole AbetaP and exhibited antiaggregating properties similar to those of antibodies obtained by injection of Abeta fibrils. Production and performance of anti-beta-amyloid antibodies in the transgenic mouse model of AD showed that these antibodies may be delivered from the periphery to the central nervous system, preventing the formation of Abeta and dissolving already-present aggregates. Moreover, immunization with Abeta protected transgenic mice from the learning and age-related memory deficits that occur in AD. These data support the hypotheses that Abeta plays a central role in AD and that site-directed antibodies that modulate Abeta conformation may provide immunotherapy of the disease.     

Vaccination with soluble Aβ oligomers generates toxicity-neutralizing antibodies

In recent studies of transgenic models of Alzheimer's disease (AD), it has been reported that antibodies to aged beta amyloid peptide 1-42 (Abeta(1-42)) solutions (mixtures of Abeta monomers, oligomers and amyloid fibrils) cause conspicuous reduction of amyloid plaques and neurological improvement. In some cases, however, neurological improvement has been independent of obvious plaque reduction, and it has been suggested that immunization might neutralize soluble, non-fibrillar forms of Abeta. It is now known that Abeta toxicity resides not only in fibrils, but also in soluble protofibrils and oligomers. The current study has investigated the immune response to low doses of Abeta(1-42) oligomers and the characteristics of the antibodies they induce. Rabbits that were injected with Abeta(1-42) solutions containing only monomers and oligomers produced antibodies that preferentially bound to assembled forms of Abeta in immunoblots and in physiological solutions. The antibodies have proven useful for assays that can detect inhibitors of oligomer formation, for immunofluorescence localization of cell-attached oligomers to receptor-like puncta, and for immunoblots that show the presence of SDS-stable oligomers in Alzheimer's brain tissue. The antibodies, moreover, were found to neutralize the toxicity of soluble oligomers in cell culture. Results support the hypothesis that immunizations of transgenic mice derive therapeutic benefit from the immuno-neutralization of soluble Abeta-derived toxins. Analogous immuno-neutralization of oligomers in humans may be a key in AD vaccines.     

Towards Alzheimer's beta-amyloid vaccination.

Beta-amyloid pathology, the main hallmark of Alzheimer's disease (AD), has been linked to its conformational status and aggregation. We recently showed that site-directed monoclonal antibodies (mAbs) towards the N-terminal region of the human beta-amyloid peptide bind to preformed beta-amyloid fibrils (Abeta), leading to disaggregation and inhibition of their neurotoxic effect. Here we report the development of a novel immunization procedure to raise effective anti-aggregating amyloid beta-protein (AbetaP) antibodies, using as antigen filamentous phages displaying the only EFRH peptide found to be the epitope of these antibodies. Due to the high antigenicity of the phage no adjuvant is required to obtain high affinity anti-aggregating IgG antibodies in animals model, that exhibit identity to human AbetaP. Such antibodies are able to sequester peripheral AbetaP, thus avoiding passage through the blood brain barrier (BBB) and, as recently shown in a transgenic mouse model, to cross the BBB and dissolve already formed beta-amyloid plaques. To our knowledge, this is the first attempt to use as a vaccine a self-anti-aggregating epitope displayed on a phage, and this may pave the way to treat abnormal accumulation-peptide diseases, such as Alzheimer's disease or other amyloidogenic diseases.     

Polypeptide conjugates comprising a beta-amyloid plaque-specific epitope as new vaccine structures against Alzheimer's disease

Immunotherapeutic approaches designed to induce a humoral immune response have recently been developed for possible vaccination to the treatment of Alzheimer's disease (AD). Based on the identification of Abeta(4-10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, branched polypeptide conjugates with this epitope peptide were synthesized and characterized. In order to produce immunogenic constructs, the Abeta(4-10) epitope alone or together with a promiscuous T-helper cell epitope peptide (FFLLTRILTIPQSLD) were attached via thioether linkage to different branched chain polymeric polypeptides with Ser or Glu in the side chains. A single peptide containing both an Abeta(4-10) and T-helper cell epitope, joined by a dipeptide Cys-Acp spacer, was also attached through the thiol function to chloroacetylated poly[Lys(Seri-DL-Alax)] (SAK). Comparative binding studies of the conjugates with a monoclonal antibody against the beta-amyloid(1-17) peptide in mice were performed by direct ELISA. The conformational preferences of carriers and conjugates in water and in a 9:1 trifluoroethanol:water mixture (v/v) was analyzed by CD spectroscopy. Experimental data showed that the chemical nature of the carrier macromolecule, and the attachment site of the epitope to the carrier, have significant effects on antibody recognition, but have no marked influence on the solution conformation of the conjugates.     

Amyloid-beta(1-42) rapidly forms protofibrils and oligomers by distinct pathways in low concentrations of sodium dodecylsulfate

Alzheimer's disease (AD) is characterized by large numbers of senile plaques in the brain that consist of fibrillar aggregates of 40- and 42-residue amyloid-beta (Abeta) peptides. However, the degree of dementia in AD correlates better with the concentration of soluble Abeta species assayed biochemically than with histologically determined plaque counts, and several investigators now propose that soluble aggregates of Abeta are the neurotoxic agents that cause memory deficits and neuronal loss. These endogenous aggregates are minor components in brain extracts from AD patients and transgenic mice that express human Abeta, but several species have been detected by gel electrophoresis in sodium dodecylsulfate (SDS) and isolated by size exclusion chromatography (SEC). Endogenous Abeta aggregation is stimulated at cellular interfaces rich in lipid rafts, and anionic micelles that promote Abeta aggregation in vitro may be good models of these interfaces. We previously found that micelles formed in dilute SDS (2 mM) promote Abeta(1-40) fiber formation by supporting peptide interaction on the surface of a single micelle complex. In contrast, here we report that monomeric Abeta(1-42) undergoes an immediate conversion to a predominant beta-structured conformation in 2 mM SDS which does not proceed to amyloid fibrils. The conformational change is instead rapidly followed by the near quantitative conversion of the 4 kDa monomer SDS gel band to 8-14 kDa bands consistent with dimers through tetramers. Removal of SDS by dialysis gave a shift in the predominant SDS gel bands to 30-60 kDa. While these oligomers resemble the endogenous aggregates, they are less stable. In particular, they do not elute as discrete species on SEC, and they are completed disaggregated by boiling in 1% SDS. It appears that endogenous oligomeric Abeta aggregates are stabilized by undefined processes that have not yet been incorporated into in vitro Abeta aggregation procedures.     

I32E and V36K double mutation in beta2-sheet abrogates amyloid beta peptide toxicity

Alzheimer's disease (AD) is the most common cause of dementia in the elderly, wherein, the accumulation of amyloid beta (Abeta) peptide as cytotoxic oligomers leads to neuropathologic changes. Transgenic mice with brain Abeta plaques immunized with aggregated Abeta have reduced amyloid burden and improved cognitive functions. However, such active immunization in humans led to a small but significant occurrence of meningoencephalitis in 6% AD volunteers due to Abeta induced toxicity. In an attempt to develop safer alternative vaccines, the design of a highly soluble peptide homologous to Abeta (Abeta-EK), that has a reduced amyloidogenic potential while maintaining the major immunogenic epitopes of Abeta is reported. More importantly, this homologue has been shown to be non-toxic, as this peptide failed to exert any observable effect on erythrocytes. The results of the present study suggests that immunization with non-toxic Abeta derivative may offer a safer therapeutic approach to AD, instead of using toxic Abeta fibrils.     

Design, structural, and immuno-analytical properties of antigenic bioconjugates comprising a beta-amyloid-plaque specific epitope

Immunotherapeutic approaches are investigated for treatment of neurodegenerative diseases of the Alzheimer's dementia (AD) type. The identification of a beta-amyloid-plaque specific epitope, Abeta(4-10) (4FRHDSGY10), recognized by therapeutically active antibodies from transgenic AD mice could provide the basis for the development of AD vaccines. Here we report on the synthesis, structural and immuno-analytical characterization of bioconjugates comprising the beta-amyloid(4-10) epitope as new vaccine lead structures against Alzheimer's disease. To produce antigenic bioconjugates, potential immunogens, the epitope peptide elongated by a cysteine residue or a cysteinyl-pentaglycine hexapeptide unit either at the N- or C-terminus was attached via a thioether bond to synthetic oligopeptide carriers, such as oligotuftsin derivatives, sequential oligopeptide carrier, or lysine dendrimer. The antigenic properties of these constructs were determined by enzyme-linked immunosorbent assay (ELISA) using an anti-Abeta(1-17) monoclonal antibody. Our results indicate that the major factors which influence the antibody binding of the Abeta(4-10) epitope are (i) the epitope topology and (ii) the presence of a spacer moiety between the carrier and the epitope peptide. Interestingly, the carrier type had no marked effect on the binding of the antibody to the epitope-conjugates. The conformational preferences of the conjugates were examined by circular dichroism spectroscopy in water and in trifluoroethanol. In water, the conjugates adopt random coil conformation independently on their primary structure. However, differences related to the attachment site of the epitope to the carriers were determined in TFE, conjugates in which the epitope was attached to the carrier through the N-terminus exhibiting more ordered secondary structure.     

The oxidative stress metabolite 4-hydroxynonenal promotes Alzheimer protofibril formation

4-Hydroxynonenal (4-HNE), formed as a consequence of oxidative stress, exists at increased concentrations in Alzheimer's disease (AD) patients and is found in amyloid beta peptide (Abeta) plaques associated with AD. Although it remains an open question as to whether oxidative stress is a causative factor or a consequence of AD, we show here that 4-HNE, putatively resulting from the peroxidation of lipids, covalently modifies Abeta, triggering its aggregation. These Abeta modifications result from 1,4 conjugate addition and/or Schiff base formation, they occur at multiple locations on a single Abeta peptide, and they result in covalent cross-linking of Abeta peptides. The consequence of these reactions is that 4-HNE accelerates the formation of Abeta protofibrils while inhibiting the production of straight, mature fibrils. Recent studies implicating Abeta oligomers and protofibrils in the neurotoxic process that ultimately leads to AD suggest that the Abeta aggregates induced by 4-HNE may be important in the pathogenesis of AD. These results provide further incentive to understand the role of oxidative stress and small-molecule Abeta modifications in sporadic AD.     

Monoclonal antibodies that target pathological assemblies of Abeta

Amyloid beta (Abeta) immunotherapy for Alzheimer's disease has shown initial success in mouse models of Alzheimer's disease and in human patients. However, because of meningoencephalitis in clinical trials of active vaccination, approaches using therapeutic antibodies may be preferred. As a novel antigen to generate monoclonal antibodies, the current study has used Abeta oligomers (amyloid beta-derived diffusible ligands, ADDLs), pathological assemblies known to accumulate in Alzheimer's disease brain. Clones were selected for the ability to discriminate Alzheimer's disease from control brains in extracts and tissue sections. These antibodies recognized Abeta oligomers and fibrils but not the physiologically prevalent Abeta monomer. Discrimination derived from an epitope found in assemblies of Abeta1-28 and ADDLs but not in other sequences, including Abeta1-40. Immunoneutralization experiments showed that toxicity and attachment of ADDLs to synapses in culture could be prevented. ADDL-induced reactive oxygen species (ROS) generation was also inhibited, establishing this response to be oligomer-dependent. Inhibition occurred whether ADDLs were prepared in vitro or obtained from Alzheimer's disease brain. As conformationally sensitive monoclonal antibodies that selectively immunoneutralize binding and function of pathological Abeta assemblies, these antibodies provide tools by which pathological Abeta assemblies from Alzheimer's disease brain might be isolated and evaluated, as well as offering a valuable prototype for new antibodies useful for Alzheimer's disease therapeutics.     

Role of aggregation conditions in structure, stability, and toxicity of intermediates in the Abeta fibril formation pathway

beta-amyloid peptide (Abeta) is one of the main protein components of senile plaques associated with Alzheimer's disease (AD). Abeta readily aggregates to forms fibrils and other aggregated species that have been shown to be toxic in a number of studies. In particular, soluble oligomeric forms are closely related to neurotoxicity. However, the relationship between neurotoxicity and the size of Abeta aggregates or oligomers is still under investigation. In this article, we show that different Abeta incubation conditions in vitro can affect the rate of Abeta fibril formation, the conformation and stability of intermediates in the aggregation pathway, and toxicity of aggregated species formed. When gently agitated, Abeta aggregates faster than Abeta prepared under quiescent conditions, forming fibrils. The morphology of fibrils formed at the end of aggregation with or without agitation, as observed in electron micrographs, is somewhat different. Interestingly, intermediates or oligomers formed during Abeta aggregation differ greatly under agitated and quiescent conditions. Unfolding studies in guanidine hydrochloride indicate that fibrils formed under quiescent conditions are more stable to unfolding in detergent than aggregation associated oligomers or Abeta fibrils formed with agitation. In addition, Abeta fibrils formed under quiescent conditions were less toxic to differentiated SH-SY5Y cells than the Abeta aggregation associated oligomers or fibrils formed with agitation. These results highlight differences between Abeta aggregation intermediates formed under different conditions and provide insight into the structure and stability of toxic Abeta oligomers.     

Synthesis, solution conformation, and antibody recognition of oligotuftsin-based conjugates containing a beta-amyloid(4-10) plaque-specific epitope

One possible therapeutic approach to treat or prevent Alzheimer's disease (AD) is immunotherapy. On the basis of the identification of Abeta(4-10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, conjugates with defined structures containing the epitope peptide attached to a tetratuftsin derivative as an oligopeptide carrier were synthesized and their structure characterized. To produce immunogenic constructs, the Abeta(4-10) epitope alone or flanked by alpha- or beta-alanine residues was attached through an amide bond to the tetratuftsin derivative (Ac-[TKPKG]4-NH2) or to a carrier peptide elongated by a promiscuous T-helper cell epitope (Ac-FFLLTRILTIPQSLD-[TKPKG]4-NH2). The conformational preferences of the carrier and conjugates were examined by CD spectroscopy in water and in 1:1 and 9:1 TFE:water mixtures (v/v). We found that the presence of flanking dimers in the conjugates had no effects on the generally unordered solution conformation of the conjugates. However, conjugates with an elongated peptide backbone exhibited CD spectra indicative for a partially ordered secondary structure in the presence of TFE. Comparative ELISA binding studies, using monoclonal antibody raised against the beta-amyloid (1-17) peptide, showed that conjugates with T-helper cell epitope in the carrier backbone exhibited decreased monoclonal antibody recognition. However, we found that this effect was compensated in conjugates comprising the Abeta(4-10) B-cell epitope with the beta-alanine dimer flanking regions at both N- and C-termini. Results suggest that modification of the B-cell epitope peptide from Abeta with rational combination of structural elements (e.g. conjugation to carrier, introduction of flanking dimers) can result in synthetic antigen with preserved antibody recognition.     

Abeta40 protects non-toxic Abeta42 monomer from aggregation

Abeta40 and Abeta42 are the predominant Abeta species in the human body. Toxic Abeta42 oligomers and fibrils are believed to play a key role in causing Alzheimer's disease (AD). However, the role of Abeta40 in AD pathogenesis is not well established. Emerging evidence indicates a protective role for Abeta40 in AD pathogenesis. Although Abeta40 is known to inhibit Abeta42 fibril formation, it is not clear whether the inhibition acts on the non-toxic monomer or acts on the toxic Abeta42 oligomers. In contrast to conventional methods that detect the appearance of fibrils, in our study Abeta42 aggregation was monitored by the decreasing NMR signals from Abeta42 monomers. In addition, differential NMR isotope labelling enabled the selective observation of Abeta42 aggregation in a mixture of Abeta42 and Abeta40. We found Abeta40 monomers inhibit the aggregation of non-toxic Abeta42 monomers, in an Abeta42/Abeta40 ratio-dependent manner. NMR titration revealed that Abeta40 monomers bind to Abeta42 aggregates with higher affinity than Abeta42 monomers. Abeta40 can also release Abeta42 monomers from Abeta42 aggregates. Thus, Abeta40 likely protects Abeta42 monomers by competing for the binding sites on pre-existing Abeta42 aggregates. Combining our data with growing evidence from transgenic mice and human genetics, we propose that Abeta40 plays a critical, protective role in Alzheimer's by inhibiting the aggregation of Abeta42 monomer. Abeta40 itself, a peptide already present in the human body, may therefore be useful for AD prevention and therapy.     

Targeting amyloid-beta peptide (Abeta) oligomers by passive immunization with a conformation-selective monoclonal antibody improves learning and memory in Abeta precursor protein (APP) transgenic mice

Passive immunization of murine models of Alzheimer disease amyloidosis reduces amyloid-beta peptide (Abeta) levels and improves cognitive function. To specifically address the role of Abeta oligomers in learning and memory, we generated a novel monoclonal antibody, NAB61, that preferentially recognizes a conformational epitope present in dimeric, small oligomeric, and higher order Abeta structures but not full-length amyloid-beta precursor protein or C-terminal amyloid-beta precursor protein fragments. NAB61 also recognized a subset of brain Abeta deposits, preferentially mature senile plaques, and amyloid angiopathy. Using NAB61 as immunotherapy, we showed that aged Tg2576 transgenic mice treated with NAB61 displayed significant improvements in spatial learning and memory relative to control mice. These data implicated Abeta oligomers as a pathologic substrate for cognitive decline in Alzheimer disease.     

Conformational HER-2/neu B-cell epitope peptide vaccine designed to incorporate two native disulfide bonds enhances tumor cell binding and antitumor activities

Cancer vaccines designed to elicit an antibody response that target antigenic sites on a tumor antigen must closely mimic the three-dimensional structure of the corresponding region on the antigen. We have designed a complex immunogen derived from the extracellular domain of human HER-2/neu-(626-649) that represents a three-dimensional epitope. We have successfully introduced two disulfide bonds into this sequence, thereby recapitulating the natural disulfide pairings observed in the native protein. To evaluate the immunogenicity of the doubly cyclized disulfide-linked peptide versus the free uncyclized peptide we examined the induction of antibody responses in both inbred and outbred mice strains, with both constructs eliciting high titered antibodies. The disulfide-paired specific antibodies exhibited enhanced cross-reactivity to HER-2/neu expressed on BT-474 cell line as determined by flow cytometry. The antitumor activities of the disulfidepaired specific antibodies did not improve the in vitro growth inhibition of human breast cancer cells overexpressing HER-2, but showed superior antitumor responses in the context of ADCC and interferon-gamma induction. Inbred mice (FVB/n) vaccinated with the disulfide-paired epitope exhibited a statistically significant reduction in the development of exogenously administered tumors in vivo compared with mice receiving either the free uncyclized or the promiscuous T-cell epitope (MVF) control peptide (p = 0.001). This study demonstrates the feasibility and importance of designing conformational epitopes that mimic the tertiary structure of the native protein for eliciting biologically relevant anti-tumor antibodies. Such approaches are a prerequisite to the design of effective peptide vaccines.     

Zinc binding agonist effect on the recognition of the beta-amyloid (4-10) epitope by anti-beta-amyloid antibodies

Amyloid plaques associated to Alzheimer's disease present a high content of zinc ions. We previously showed that the N-terminal region of the amyloid peptide Abeta constitutes an autonomous zinc-binding domain. This region encompasses the previously identified epitope Abeta(4-10) targeted by antibodies capable to reduce amyloid deposition, but the influence of Abeta/Zn binding on the epitope recognition remains unknown. We demonstrate here the effect of Zn2+ ions on the recognition of peptides sharing the sequence of the Abeta N-terminal domain, by two monoclonal antibodies recognizing the beta-amyloid(4-10) epitope. The presence of Zn2+, but not of other cations, increased the recognition of the (1-16) peptide, while it was without effect on the recognition of the (1-10) peptide. These findings show a zinc-induced conformational change of the (1-16)-N-terminal region of AP3, which results in a better accessibility of the Abeta(4-10) epitope to the anti-Abeta antibodies, and suggest a role of zinc in epitope-based vaccination approaches.     

Structural studies of Alzheimer's disease Abeta peptide oligomers

Amyloid beta peptide is recognized as the main constituent of the extracellular amyloid plaques, the major neuropathological hallmark of Alzheimer's disease. Abeta is a small peptide constitutively expressed in normal cells, not toxic in the monomeric form but aggregated Abeta is assumed to be the main if not the only factor causing Alzheimer's disease. Interestingly, the new reports suggest neurotoxicity of soluble Abeta oligomers rather than amyloid fibrils. Because of the fact that fibrils were thought to be the main toxic species in AD, early structural studies focused on fibrils themselves and Abeta monomers, as their building blocks while there is practically no data on oligomer structure and mechanism of neurotoxicity. Using a model peptide spanning residues 10–30 of Abeta, obtained by overexpression in bacteria, we have employed mass spectrometry of noncovalent complexes and disulfide rearrangement assay to gain new insight into structure and dynamics of a prenucleation step of Abeta peptide oligomerisation.     

Prototype Alzheimer's disease vaccine using the immunodominant B cell epitope from beta-amyloid and promiscuous T cell epitope pan HLA DR-binding peptide

Immunization of amyloid precursor protein transgenic mice with fibrillar beta-amyloid (Abeta) prevents Alzheimer's disease (AD)-like neuropathology. The first immunotherapy clinical trial used fibrillar Abeta, containing the B and T cell self epitopes of Abeta, as the immunogen formulated with QS21 as the adjuvant in the vaccine. Unfortunately, the clinical trial was halted during the phase II stage when 6% of the participants developed meningoencephalitis. The cause of the meningoencephalitis in the patients that received the vaccine has not been definitively determined; however, analysis of two case reports from the AN-1792 vaccine trial suggest that the meningoencephalitis may have been caused by a T cell-mediated autoimmune response, whereas production of anti-Abeta Abs may have been therapeutic to the AD patients. Therefore, to reduce the risk of an adverse T cell-mediated immune response to Abeta immunotherapy we have designed a prototype epitope vaccine that contains the immunodominant B cell epitope of Abeta in tandem with the synthetic universal Th cell pan HLA DR epitope, pan HLA DR-binding peptide (PADRE). Importantly, the PADRE-Abeta(1-15) sequence lacks the T cell epitope of Abeta. Immunization of BALB/c mice with the PADRE-Abeta(1-15) epitope vaccine produced high titers of anti-Abeta Abs. Splenocytes from immunized mice showed robust T cell stimulation in response to peptides containing PADRE. However, splenocytes from immunized mice were not reactivated by the Abeta peptide. New preclinical trials in amyloid precursor protein transgenic mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse events that occurred in the first clinical trial.     

Mitochondrial Ca2+ overload underlies Abeta oligomers neurotoxicity providing an unexpected mechanism of neuroprotection by NSAIDs

Dysregulation of intracellular Ca(2+) homeostasis may underlie amyloid beta peptide (Abeta) toxicity in Alzheimer's Disease (AD) but the mechanism is unknown. In search for this mechanism we found that Abeta(1-42) oligomers, the assembly state correlating best with cognitive decline in AD, but not Abeta fibrils, induce a massive entry of Ca(2+) in neurons and promote mitochondrial Ca(2+) overload as shown by bioluminescence imaging of targeted aequorin in individual neurons. Abeta oligomers induce also mitochondrial permeability transition, cytochrome c release, apoptosis and cell death. Mitochondrial depolarization prevents mitochondrial Ca(2+) overload, cytochrome c release and cell death. In addition, we found that a series of non-steroidal anti-inflammatory drugs (NSAIDs) including salicylate, sulindac sulfide, indomethacin, ibuprofen and R-flurbiprofen depolarize mitochondria and inhibit mitochondrial Ca(2+) overload, cytochrome c release and cell death induced by Abeta oligomers. Our results indicate that i) mitochondrial Ca(2+) overload underlies the neurotoxicity induced by Abeta oligomers and ii) inhibition of mitochondrial Ca(2+) overload provides a novel mechanism of neuroprotection by NSAIDs against Abeta oligomers and AD.     

Human plasma contains cross-reactive Abeta conformer-specific IgG antibodies

Two conformers of aggregated Abeta, i.e., fibrils and oligomers, have been deemed important in the pathogenesis of Alzheimer's disease. We now report that intravenous immune globulin (IVIG) derived from pools of human plasma contains IgGs that recognize conformational epitopes present on fibrils and oligomers, but not their soluble monomeric precursor. We have used affinity chromatography to isolate these antibodies and have shown that they cross-reacted with comparable nanomolar avidity with both types of Abeta aggregates; notably, binding was not inhibited by soluble Abeta monomers. Our studies provide further support for investigating the therapeutic use of IVIG in Alzheimer's disease.