Central repeat fragment of reelin leads to active reelin intracellular signaling and rescues cognitive deficits in a mouse model of reelin deficiency.

Reelin and its receptor, ApoER2, play important roles in prenatal brain development and postnatally in synaptic plasticity, learning, and memory. Previous reports suggest that reelin's central fragment binds to ApoER2 and receptor clustering is involved in subsequent intracellular signaling. However, limitations of currently available assays have not established cellular evidence of ApoER2 clustering upon binding of the central reelin fragment. In the present study, we developed a novel, cell-based assay of ApoER2 dimerization using a "split-luciferase" approach. Specifically, cells were co-transfected with one recombinant ApoER2 receptor fused to the N-terminus of luciferase and one ApoER2 receptor fused to the C-terminus of luciferase. Using this assay, we directly observed basal ApoER2 dimerization/clustering in transfected HEK293T cells and, significantly, an increase in ApoER2 clustering in response to that central fragment of reelin. Furthermore, the central fragment of reelin activated intracellular signal transduction of ApoER2, indicated by increased levels of phosphorylation of Dab1, ERK1/2, and Akt in primary cortical neurons. Functionally, we were able to demonstrate that injection of the central fragment of reelin rescued phenotypic deficits observed in the heterozygous reeler mouse. These data are the first to test the hypothesis that the central fragment of reelin contributes to facilitating the reelin intracellular signaling pathway through receptor clustering.

A novel Reelin construct, R36, recovered behavioural deficits in the heterozygous reeler mouse.

Reelin, a large extracellular glycoprotein, plays a critical role in prenatal brain development and postnatally in synaptic plasticity, learning and memory. Dysregulation of Reelin signalling has been implicated in several neuropsychiatric disorders including schizophrenia, autism, depression and Alzheimer's disease. Previous studies have demonstrated that Reelin's central fragment, R3456, binds to ApoER2, inducing ApoER2 clustering and subsequent intracellular signalling. We previously reported the development of a novel luciferase complementation assay, which we used to demonstrate that R3456 can lead to ApoER2 receptor dimerization. Using this same assay, we explored various smaller fragments and combinations from R3456, and we identified a construct of repeats 3 and 6 (R36), which could still elicit equivalent receptor dimerization. The purpose of this study was to test R36 for biological effects in vitro and in vivo. We show that R36 was capable of initiating intracellular signalling in primary neuronal cultures. In addition, we demonstrate that a single intracerebroventricular injection of R36 protein into a model of Reelin deficiency, the heterozygous reeler mice, can significantly improve cognition. These data support a role for the new construct R36 to enhance the Reelin pathway, and the future possibility of exploring gene therapy approaches with R36 in diseases characterized by reduced levels of Reelin.

Concentration and proteolysis of CX3CL1 may regulate the microglial response to CX3CL1.

Fractalkine (FKN) is a membrane-bound chemokine that can be cleaved by proteases such as ADAM 10, ADAM 17, and cathepsin S to generate soluble fragments. Studies using different forms of the soluble FKN yield conflicting results in vivo. These observations prompted us to investigate the function and pharmacology of two commonly used isoforms of FKN, a human full-length soluble FKN (sFKN), and a human chemokine domain only FKN (cdFKN). Both are prevalent in the literature and are often assumed to be functionally equivalent. We observed that recombinant sFKN and cdFKN exhibit similar potencies in a cell-based cAMP assay, but binding affinity for CX3CR1 was modestly different. There was a 10-fold difference in potency between sFKN and cdFKN when assessing their ability to stimulate ß-arrestin recruitment. Interestingly, high concentrations of FKN, regardless of cleavage variant, were ineffective at reducing pro-inflammatory microglial activation and may induce a pro-inflammatory response. This effect was observed in mouse and rat primary microglial cells as well as microglial cell lines. The inflammatory response was exacerbated in aged microglia, which is known to exhibit age-related inflammatory phenotypes. We observed the same effects in Cx3cr1-/- primary microglia and therefore speculate that an alternative FKN receptor may exist. Collectively, these data provide greater insights into the function and pharmacology of these common FKN reagents, which may clarify conflicting reports and urge greater caution in the selection of FKN peptides for use in in vitro and in vivo studies and the interpretation of results obtained using these differing peptides.

Reelin central fragment supplementation improves cognitive deficits in a mouse model of Fragile X Syndrome.

Fragile X Syndrome (FXS) is the most common form of inherited intellectual disability and is characterized by autistic behaviors, childhood seizures, and deficits in learning and memory. FXS has a loss of function of the FMR1 gene that leads to a lack of Fragile X Mental Retardation Protein (FMRP) expression. FMRP is critical for synaptic plasticity, spatial learning, and memory. Reelin is a large extracellular glycoprotein essential for synaptic plasticity and numerous neurodevelopmental processes. Reduction in Reelin signaling is implicated as a contributing factor in disease etiology in several neurological disorders, including schizophrenia, and autism. However, the role of Reelin in FXS is poorly understood. We demonstrate a reduction in Reelin in Fmr1 knock-out (KO) mice, suggesting that a loss of Reelin activity may contribute to FXS. We demonstrate here that Reelin signaling enhancement via a single intracerebroventricular injection of the Reelin central fragment into Fmr1 KO mice can profoundly rescue cognitive deficits in hidden platform water maze and fear conditioning, as well as hyperactivity during the open field. Improvements in behavior were associated with rescued levels of post synaptic marker in Fmr1 KO mice when compared to controls. These data suggest that increasing Reelin signaling in FXS could offer a novel therapeutic for improving cognition in FXS.

Intraventricular human immunoglobulin distributes extensively but fails to modify amyloid in a mouse model of amyloid deposition.

Intravenous immunoglobulin infusions into Alzheimer patients have been found to provide cognitive benefit over a period of 6 mo in open label studies. One suggestion has been that these preparations contain small amounts of antibodies directed against monomeric and oligomeric Aß which underlie their effectiveness in patients. To test this hypothesis, we infused Gammagard, a version of intravenous immunoglobulin (IVIG), into the lateral ventricle of amyloid precursor protein (APP) transgenic mice with pre-existing amyloid deposits. Mice were infused over 4 weeks, and tested behaviorally for the last 2 weeks of treatment. Brains were analyzed for histopathology. We found widespread distribution of human-immunoglobulin G (h-IgG) staining in the mouse forebrain, including cerebral cortices and hippocampus. Some cortical neurons appeared to concentrate the h-IgG, but we did not detect evidence of amyloid plaque labeling by h-IgG. The IVIG-treated mice had no change in phenotype compared to saline-infused animals with respect to activity, learning and memory, or amyloid deposition. APP mice infused with an anti-Aß monoclonal antibody did show some reduction in amyloid deposits. These data do not support the argument that anti-Aß antibodies in IVIG preparations are responsible for cognitive benefits seen with these preparations.

Tau accumulation activates the unfolded protein response by impairing endoplasmic reticulum-associated degradation.

In Alzheimer's disease (AD), the mechanisms of neuronal loss remain largely unknown. Although tau pathology is closely correlated with neuronal loss, how its accumulation may lead to activation of neurotoxic pathways is unclear. Here we show that tau increased the levels of ubiquitinated proteins in the brain and triggered activation of the unfolded protein response (UPR). This suggested that tau interferes with protein quality control in the endoplasmic reticulum (ER). Consistent with this, ubiquitin was found to associate with the ER in human AD brains and tau transgenic (rTg4510) mouse brains, but this was not always colocalized with tau. The increased levels of ubiquitinated protein were accompanied by increased levels of phosphorylated protein kinase R-like ER kinase (pPERK), a marker that indicates UPR activation. Depleting soluble tau levels in cells and brain could reverse UPR activation. Tau accumulation facilitated its deleterious interaction with ER membrane and associated proteins that are essential for ER-associated degradation (ERAD), including valosin-containing protein (VCP) and Hrd1. Based on this, the effects of tau accumulation on ERAD efficiency were evaluated using the CD3δ reporter, an ERAD substrate. Indeed, CD3δ accumulated in both in vitro and in vivo models of tau overexpression and AD brains. These data suggest that soluble tau impairs ERAD and the result is activation of the UPR. The reversibility of this process, however, suggests that tau-based therapeutics could significantly delay this type of cell death and therefore disease progression.

Reelin supplementation recovers sensorimotor gating, synaptic plasticity and associative learning deficits in the heterozygous reeler mouse.

The lipoprotein receptor ligand Reelin is important for the processes of normal synaptic plasticity, dendritic morphogenesis, and learning and memory. Heterozygous reeler mice (HRM) show many neuroanatomical, biochemical, and behavioral features that are associated with schizophrenia. HRM show subtle morphological defects including reductions in dendritic spine density, altered synaptic plasticity and behavioral deficits in associative learning and memory and pre-pulse inhibition. The present studies test the hypothesis that in vivo elevation of Reelin levels can rescue synaptic and behavioral phenotypes associated with HRM. We demonstrate that a single in vivo injection of Reelin increases GAD67 expression and alters dendritic spine morphology. In parallel we observed enhancement of hippocampal synaptic function and associative learning and memory. Reelin supplementation also increases pre-pulse inhibition. These results suggest that characteristics of HRM, similar to those observed in schizophrenia, are sensitive to Reelin levels and can be modified with Reelin supplementation in male and female adults.

Chronological age impacts immunotherapy and monocyte uptake independent of amyloid load.

One vexing issue in biomedical research is the failure of many therapies to translate from success in animal models to effective treatment of human disease. One significant difference between the animal models and the human disease is the age of the subject. Cancer, stroke and Alzheimer's occur mainly in humans beyond the 75% mean survival age, while most mouse models use juvenile or young adult animals. Here we compare two mouse models of amyloid deposition, the Tg2576 APP model and the more aggressive APP+PS1 model in which a mutant presenilin1 gene is overexpressed with Tg2576. Middle-aged APP+PS1 mice and aged APP mice have similar degrees of amyloid pathology with a few differences that may partially explain some of the differences between the two age cohorts. The first study evaluated production of microhemorrhage by a monoclonal anti-Aß antibody. We found that in spite of greater amyloid clearance in middle-aged APP+PS1 mice than aged APP mice, the microhemorrhage only developed in old animals. This argues that preclinical studies of immunotherapy in young or middle-aged mice may not predict this potential liability in clinical trials. A second study evaluated the infiltration of systemically injected GFP labeled monocytes into the CNS. Here we find that infiltration is greater in aged mice than middle-aged mice, in spite of greater total Aß staining in the middle-aged animals. We conclude that preclinical studies should be conducted in aged animal models as well as young mice to better prepare for unintended consequences in the human trial.

The Hsp90 kinase co-chaperone Cdc37 regulates tau stability and phosphorylation dynamics.

The microtubule-associated protein tau, which becomes hyperphosphorylated and pathologically aggregates in a number of these diseases, is extremely sensitive to manipulations of chaperone signaling. For example, Hsp90 inhibitors can reduce the levels of tau in transgenic mouse models of tauopathy. Because of this, we hypothesized that a number of Hsp90 accessory proteins, termed co-chaperones, could also affect tau stability. Perhaps by identifying these co-chaperones, new therapeutics could be designed to specifically target these proteins and facilitate tau clearance. Here, we report that the co-chaperone Cdc37 can regulate aspects of tau pathogenesis. We found that suppression of Cdc37 destabilized tau, leading to its clearance, whereas Cdc37 overexpression preserved tau. Cdc37 was found to co-localize with tau in neuronal cells and to physically interact with tau from human brain. Moreover, Cdc37 levels significantly increased with age. Cdc37 knockdown altered the phosphorylation profile of tau, an effect that was due in part to reduced tau kinase stability, specifically Cdk5 and Akt. Conversely, GSK3ß and Mark2 were unaffected by Cdc37 modulation. Cdc37 overexpression prevented whereas Cdc37 suppression potentiated tau clearance following Hsp90 inhibition. Thus, Cdc37 can regulate tau in two ways: by directly stabilizing it via Hsp90 and by regulating the stability of distinct tau kinases. We propose that changes in the neuronal levels or activity of Cdc37 could dramatically alter the kinome, leading to profound changes in the tau phosphorylation signature, altering its proteotoxicity and stability.

Phenothiazine-mediated rescue of cognition in tau transgenic mice requires neuroprotection and reduced soluble tau burden.

BACKGROUND: It has traditionally been thought that the pathological accumulation of tau in Alzheimer's disease and other tauopathies facilitates neurodegeneration, which in turn leads to cognitive impairment. However, recent evidence suggests that tau tangles are not the entity responsible for memory loss, rather it is an intermediate tau species that disrupts neuronal function. Thus, efforts to discover therapeutics for tauopathies emphasize soluble tau reductions as well as neuroprotection. RESULTS: Here, we found that neuroprotection alone caused by methylene blue (MB), the parent compound of the anti-tau phenothiaziazine drug, Rember™, was insufficient to rescue cognition in a mouse model of the human tauopathy, progressive supranuclear palsy (PSP) and fronto-temporal dementia with parkinsonism linked to chromosome 17 (FTDP17): Only when levels of soluble tau protein were concomitantly reduced by a very high concentration of MB, was cognitive improvement observed. Thus, neurodegeneration can be decoupled from tau accumulation, but phenotypic improvement is only possible when soluble tau levels are also reduced. CONCLUSIONS: Neuroprotection alone is not sufficient to rescue tau-induced memory loss in a transgenic mouse model. Development of neuroprotective agents is an area of intense investigation in the tauopathy drug discovery field. This may ultimately be an unsuccessful approach if soluble toxic tau intermediates are not also reduced. Thus, MB and related compounds, despite their pleiotropic nature, may be the proverbial "magic bullet" because they not only are neuroprotective, but are also able to facilitate soluble tau clearance. Moreover, this shows that neuroprotection is possible without reducing tau levels. This indicates that there is a definitive molecular link between tau and cell death cascades that can be disrupted.

Trafficking CD11b-positive blood cells deliver therapeutic genes to the brain of amyloid-depositing transgenic mice.

A major question for gene therapy in brain concerns methods to administer therapeutic genes in a uniform manner over major portions of the brain. A second question in neuroimmunology concerns the extent to which monocytes migrate to the CNS in degenerative disorders. Here we show that CD11b+ cells (largely monocytes) isolated from the bone marrow of GFP (green fluorescent protein)-expressing donors spontaneously home to compacted amyloid plaques in the brain. Injections of these cells as a single pulse show a rapid clearance from circulation (90 min half-life) and tissue residence half-lives of approximately 3 d. The uptake into brain was minimal in nontransgenic mice. In transgenic mice containing amyloid deposits, uptake was dramatically increased and associated with a corresponding decrease in monocyte uptake into peripheral organs compared to nontransgenic littermates. Twice weekly infusions of the CD11b+ bone marrow cells transfected with a genetically engineered form of the protease neprilysin completely arrest amyloid deposition in an aggressively depositing transgenic model. Exploiting the natural homing properties of peripherally derived blood cells to deliver therapeutic genes has the advantages of access to the entire CNS, expression largely restricted to sites of injury, low risk of immune reactivity, and fading of expression if adverse reactions are encountered. These observations support the feasibility of testing autologous monocytes for application of therapeutic genes in human CNS disease. Moreover, these data support the results from bone marrow grafts that circulating CD11b+ cells can enter the CNS without requiring the use of lethal irradiation.

Hsc70 rapidly engages tau after microtubule destabilization.

The microtubule-associated protein Tau plays a crucial role in regulating the dynamic stability of microtubules during neuronal development and synaptic transmission. In a group of neurodegenerative diseases, such as Alzheimer disease and other tauopathies, conformational changes in Tau are associated with the initial stages of disease pathology. Folding of Tau into the MC1 conformation, where the amino acids at residues 7-9 interact with residues 312-342, is one of the earliest pathological alterations of Tau in Alzheimer disease. The mechanism of this conformational change in Tau and the subsequent effect on function and association to microtubules is largely unknown. Recent work by our group and others suggests that members of the Hsp70 family play a significant role in Tau regulation. Our new findings suggest that heat shock cognate (Hsc) 70 facilitates Tau-mediated microtubule polymerization. The association of Hsc70 with Tau was rapidly enhanced following treatment with microtubule-destabilizing agents. The fate of Tau released from the microtubule was found to be dependent on ATPase activity of Hsc70. Microtubule destabilization also rapidly increased the MC1 folded conformation of Tau. An in vitro assay suggests that Hsc70 facilitates formation of MC1 Tau. However, in a hyperphosphorylating environment, the formation of MC1 was abrogated, but Hsc70 binding to Tau was enhanced. Thus, under normal circumstances, MC1 formation may be a protective conformation facilitated by Hsc70. However, in a diseased environment, Hsc70 may preserve Tau in a more unstructured state, perhaps facilitating its pathogenicity.

Virus-like peptide vaccines against Abeta N-terminal or C-terminal domains reduce amyloid deposition in APP transgenic mice without addition of adjuvant.

Immunotherapy against the Abeta peptide is increasingly viewed as an effective means of preventing and even decreasing Abeta deposition in transgenic mouse models and human cases of Alzheimer's disease. A prior active immunization trial was halted due to adverse events which occurred subsequent to a change in the adjuvant used in the vaccine preparation. Although widely used in experimental studies, adjuvants available for use in vaccines intended for humans are limited. We compared two vaccine preparations in which an immunogenic bacteriophage was conjugated with either an N-terminal (Abeta1-9) or C-terminal (Abeta28-40) peptide sequence from the Abeta molecule. We found that both produced significant antibody titers without use of additional adjuvants. Surprisingly, the response to the N terminal sequence was comprised largely of a stable IgM response, while the C-terminal vaccine produced an IgG response with minimal IgM reactivity. Both of these immunogens reduced Abeta levels when tissues were examined 8 months after the first inoculation. These data demonstrate that (a) C-terminal specific vaccines can effectively lower Abeta and (b) IgM antibodies against Abeta may be capable of lowering Abeta, possibly through action in the brain rather than the periphery.

Suppression of amyloid deposition leads to long-term reductions in Alzheimer's pathologies in Tg2576 mice.

In amyloid precursor protein (APP) models of amyloid deposition, the amount of amyloid deposits increase with mouse age. At a first approximation, the extent of amyloid accumulation may either reflect small excesses of production over clearance that accumulate over time or, alternatively, indicate a steady-state equilibrium at that age, reflecting the instantaneous excess of production over clearance, which increases as the organism ages. To discriminate between these options, we reversibly suppressed amyloid deposition in Tg2576 mice with the anti-Abeta antibody 2H6, starting at 8 months, just before the first histological deposits can be discerned. Six months later, we stopped the suppression and monitored the progression of amyloid accumulation in control APP mice and suppressed APP mice over the next 3 months. The accumulation hypothesis would predict that the rate of amyloid from 14 to 17 months would be similar in the suppressed and control mice, while the equilibrium hypothesis would predict that the increase would be faster in the suppressed group, possibly catching up completely with the control mice. The results strongly support the accumulation hypothesis, with no evidence of the suppressed mice catching up with the control mice as predicted by equilibrium models. If anything, there was a slower rate of increase in the suppressed APP mice than the control mice, suggesting that a slow seeding mechanism likely precedes a rapid fibrillogenesis in determining the extent of amyloid deposition.

Contrasting in vivo effects of two peptide-based amyloid-beta protein aggregation inhibitors in a transgenic mouse model of amyloid deposition.

Previous studies have shown that 17,19,21-tri-N-methyl-Abeta16-22 peptide (Abeta16-22m), and a peptide analogue containing alpha,alpha-disubstituted amino acids (alphaalpha AA) in the hydrophobic core domain of Abeta, termed AMY-1, effectively inhibited full-length Abeta aggregation in vitro. To investigate the amyloid-modifying effects of these agents in vivo, we injected these compounds into the hippocampus of 13-month-old amyloid precursor protein (APP) transgenic mice, a model of amyloid deposition. After 7 days, brain tissues were stained for immunohistochemistry to detect total Abeta and thioflavine-S (THIO-S) to measure Abeta compact plaques. Both diffuse Abeta deposits and compact amyloid plaques were significantly increased when injecting 0.3 nmol Abeta16-22m compared to the PBS vehicle. The amyloid aggregation-modifying peptide AMY-1 showed a slight reduction of Abeta deposition in the injection area at a dose of 0.3 nmol, but neuronal toxicity, measured by Fluoro-Jade and Nissl stains, appeared when higher doses (3 nmol) were tested. Our data indicate that, unlike observations reported in vitro, the Abeta16-22m increased deposition of Abeta in the brain of APP transgenic mice in vivo. Possible explanations for this outcome include unique influences of the brain environment and/or modification of Abeta production or clearance by the administered agent. The AMY-1 peptide showed a trend for reducing Abeta deposits, but led to toxicity at higher doses. These data emphasize the need for evaluating potential Abeta aggregation inhibitors with in vivo models of amyloid deposition before assuming they will have benefit in treating Alzheimer's disease patients.

Improvement of a low pH antigen-antibody dissociation procedure for ELISA measurement of circulating anti-Abeta antibodies.

BACKGROUND: Prior work from our group found that acid dissociation (pH 2.5 incubation) of serum from APP transgenic mice vaccinated against Abeta increased the apparent anti-Abeta titers, suggesting antibody masking by antigen in the ELISA assay. Subsequently, we found that pH 2.5 incubation of serum from unvaccinated non-transgenic mice showed antibody binding to Abeta1-42, but no increase when other proteins, including shorter Abeta peptides, coated the ELISA plate. To investigate further the effects of low pH incubation on apparent anti-Abeta1-42 signals, we examined normal sera from nonTg unvaccinated mice, nonTg mice vaccinated with Abeta peptide (to produce authentic anti-Abeta antibodies) or a monoclonal antibody against Abeta (6E10) using competitive-inhibition ELISA and Abeta epitope mapping assays. In addition, we examined use of a less stringent low pH procedure at pH 3.5, to ascertain if it had the same effects as the pH 2.5 procedure. RESULTS: We believe there are three distinct effects of pH 2.5 incubation.; A) an artifactual increase in binding to full length Abeta by mouse immunoglobulin which has low affinity for Abeta, B) an inactivation of anti-Abeta antibodies that is time dependent and C) unmasking of high affinity anti-Abeta antibodies when high levels of circulating Abeta is present in APP transgenic mice. All three reactions can interact to produce the final ELISA signal. Incubation of sera from unvaccinated nonTg mice at pH 2.5 enhanced ELISA signals by process A. Conversely, pH 2.5 incubation of sera from vaccinated nonTg mice with caused a time dependent reduction of antibody signal by process B (overcoming the increase caused by A). The artifactual anti-Abeta ELISA signal enhanced by pH 2.5 incubation of normal mouse sera could not be effectively competed by low to moderate concentrations of Abeta, nor bind to shorter Abeta peptides in a manner similar to authentic anti-Abeta antibodies. Incubation of mouse sera at pH 3.5 caused neither an apparent increase in anti-Abeta ELISA signal, nor an inactivation of the ELISA signals resulting from either vaccination or monoclonal antibodies. However, incubation at pH 3.5 was able to completely reverse the reduction in ELISA signal caused by Abeta complexing with antibodies in sera from vaccinated mice or monoclonal anti-Abeta antibodies. CONCLUSION: Incubation at pH 3.5 is sufficient to dissociate Abeta bound to anti-Abeta antibodies without producing artifactual increases in the signal, or inactivating authentic antibody binding. Thus, use of pH 3.5 is a considerable improvement over pH 2.5 incubation for unmasking anti-Abeta antibodies in ELISA assays to measure antibodies in APP transgenic mouse sera.

Activation of NADPH oxidase and extracellular superoxide production in seizure-induced hippocampal damage.

We sought to determine whether the extracellular compartment contributed to seizure-induced superoxide (O2*-) production and to determine the role of the NADPH oxidase complex as a source of this O2*- production. The translocation of NADPH oxidase subunits (p47phox, p67phox and rac1) was assessed by immunoblot analysis and NADPH-driven O2*- production was measured using 2-(4-hydroxybenzyl)-6-(4-hydroxyphenyl)-8-benzyl-3,7-dihydroimidazo [1,2-alpha] pyrazin-3-one-enhanced chemiluminescence. Kainate-induced status epilepticus resulted in a time-dependent translocation of NADPH oxidase subunits (p47phox, p67phox and rac-1) from hippocampal cytosol to membrane fractions. Hippocampal membrane fractions from kainate-injected rats showed increased NADPH-driven and diphenylene iodonium-sensitive O2*- production in comparison to vehicle-treated rats. The time-course of kainate-induced NADPH oxidase activation coincided with microglial activation in the rat hippocampus. Finally, kainate-induced neuronal damage and membrane oxygen consumption were inhibited in mice overexpressing extracellular superoxide dismutase. These results suggest that seizure activity activates the membrane NADPH oxidase complex resulting in increased formation of O2*-.

Overcoming antigen masking of anti-amyloidbeta antibodies reveals breaking of B cell tolerance by virus-like particles in amyloidbeta immunized amyloid precursor protein transgenic mice.

BACKGROUND: In prior work we detected reduced anti-Abeta antibody titers in Abeta-vaccinated transgenic mice expressing the human amyloid precursor protein (APP) compared to nontransgenic littermates. We investigated this observation further by vaccinating APP and nontransgenic mice with either the wild-type human Abeta peptide, an Abeta peptide containing the "Dutch Mutation", E22Q, or a wild-type Abeta peptide conjugated to papillomavirus virus-like particles (VLPs). RESULTS: Anti-Abeta antibody titers were lower in vaccinated APP than nontransgenic mice even when vaccinated with the highly immunogenic Abeta E22Q. One concern was that human Abeta derived from the APP transgene might mask anti-Abeta antibodies in APP mice. To test this possibility, we dissociated antigen-antibody complexes by incubation at low pH. The low pH incubation increased the anti-Abeta antibody titers 20-40 fold in APP mice but had no effect in sera from nontransgenic mice. However, even after dissociation, the anti-Abeta titers were still lower in transgenic mice vaccinated with wild-type Abeta or E22Q Abeta relative to non-transgenic mice. Importantly, the dissociated anti-Abeta titers were equivalent in nontransgenic and APP mice after VLP-based vaccination. Control experiments demonstrated that after acid-dissociation, the increased antibody titer did not cross react with bovine serum albumin nor alpha-synuclein, and addition of Abeta back to the dissociated serum blocked the increase in antibody titers. CONCLUSIONS: Circulating human Abeta can interfere with ELISA assay measurements of anti-Abeta titers. The E22Q Abeta peptide vaccine is more immunogenic than the wild-type peptide. Unlike peptide vaccines, VLP-based vaccines against Abeta abrogate the effects of Abeta self-tolerance.