Ulrich Ebert

Globular amyloid β-peptide1−42 oligomer − a homogenous and stable neuropathological protein in Alzheimer's disease

Amyloid β‐peptide (Aβ)1−42 oligomers have recently been discussed as intermediate toxic species in Alzheimers disease (AD) pathology. Here we describe a new and highly stable Aβ1−42 oligomer species which can easily be prepared in vitro and is present in the brains of patients with AD and Aβ1−42‐overproducing transgenic mice. Physicochemical characterization reveals a pure, highly water‐soluble globular 60‐kDa oligomer which we named ‘Aβ1−42 globulomer’. Our data indicate that Aβ1−42 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 Aβ1−42 globulomer‐specific antibodies that do not cross‐react with amyloid precursor protein, Aβ1−40 and Aβ1−42 monomers and Aβ fibrils. Aβ1−42 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 Aβ1−42 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 Aβ globulomer structure epitope is expected to have a high potential for treatment of AD.

Amyloid β Oligomers (Aβ1–42 Globulomer) Suppress Spontaneous Synaptic Activity by Inhibition of P/Q-Type Calcium Currents

Abnormal accumulation of soluble oligomers of amyloid β (Aβ) is believed to cause malfunctioning of neurons in Alzheimers disease. It has been shown that Aβ oligomers impair synaptic plasticity, thereby altering the ability of the neuron to store information. We examined the underlying cellular mechanism of Aβ oligomer-induced synaptic modifications by using a recently described stable oligomeric Aβ preparation called “Aβ1–42 globulomer.” Synthetically prepared Aβ1–42 globulomer has been shown to localize to neurons and impairs long-term potentiation (Barghorn et al., 2005). Here, we demonstrate that Aβ1–42 globulomer does not affect intrinsic neuronal properties, as assessed by measuring input resistance and discharge characteristics, excluding an unspecific alteration of membrane properties. We provide evidence that Aβ1–42 globulomer, at concentrations as low as 8 nm, specifically suppresses spontaneous synaptic activity resulting from a reduction of vesicular release at terminals of both GABAergic and glutamatergic synapses. EPSCs and IPSCs were primarily unaffected. A detailed search for the precise molecular target of Aβ1–42 globulomer revealed a specific inhibition of presynaptic P/Q calcium currents, whereas other voltage-activated calcium currents remained unaltered. Because intact P/Q calcium currents are needed for synaptic plasticity, the disruption of such currents by Aβ1–42 globulomer may cause deficits in cellular mechanisms of information storage in brains of Alzheimers disease patients. The inhibitory effect of Aβ1–42 globulomer on synaptic vesicle release could be reversed by roscovitine, a specific enhancer of P/Q currents. Selective enhancement of the P/Q calcium current may provide a promising strategy in the treatment of Alzheimers disease.

Reduced Spine Density in Specific Regions of CA1 Pyramidal Neurons in Two Transgenic Mouse Models of Alzheimer's Disease

One major hallmark of Alzheimers disease (AD) is the massive loss of synapses that occurs at an early clinical stage of the disease. In this study, we characterize alterations in spine density and the expression of synapse-associated immediate early gene Arc (activity-regulated cytoskeleton-associated protein) in the hippocampal CA1 regions of two different amyloid precursor protein (APP) transgenic mouse lines before plaque development and their connection to performance in hippocampus-dependent memory tests. The density of mushroom-type spines was reduced by 34% in the basal dendrites proximal to the soma of CA1 pyramidal neurons in 5.5-month-old Tg2576 mice, carrying the Swedish mutation, compared with wild-type littermates. A similar reduction of 42% was confirmed in the same region of 8-month-old APP/Lo mice, carrying the London mutation. In this strain, the reduction extended to the distal dendritic spines (28%), although no differences were found in apical dendrites in either transgenic mouse line. Both transgenic mice lines presented a significant increase in Arc protein expression in CA1 compared with controls, suggesting rather an overactivity and increased spine turnover that was supported by a significant decrease in number of somatostatin-immunopositive inhibitory interneurons in the stratum oriens of CA1. Behaviorally, the transgenic mice showed decrease freezing in the fear contextual conditioning test and impairment in spatial memory assessed by Morris water maze test. These data indicate that cognitive impairment in APP transgenic mice is correlated with impairment of synaptic connectivity in hippocampal CA1, probably attributable to loss of inhibitory interneurons and subsequent hyperactivity.

Generation and Therapeutic Efficacy of Highly Oligomer-Specific β-Amyloid Antibodies

Oligomers of the β-amyloid (Aβ) peptide have been indicated in early neuropathologic changes in Alzheimers disease. Here, we present a synthetic Aβ20-42 oligomer (named globulomer) with a different conformation to monomeric and fibrillar Aβ peptide, enabling the generation of highly Aβ oligomer-specific monoclonal antibodies. The globulomer-derived antibodies specifically detect oligomeric but not monomeric or fibrillar Aβ in various Aβ preparations. The globulomer-specific antibody A-887755 was able to prevent Aβ oligomer binding and dynamin cleavage in primary hippocampal neurons and to reverse globulomer-induced reduced synaptic transmission. In amyloid precursor protein (APP) transgenic mice, vaccination with Aβ globulomer and treatment with A-887755 improved novel object recognition. The cognitive improvement is likely attributable to reversing a deficit in hippocampal synaptic spine density in APP transgenic mice as observed after treatment with A-887755. Our findings demonstrate that selective reduction of Aβ oligomers by immunotherapy is sufficient to normalize cognitive behavior and synaptic deficits in APP transgenic mice.

Characterization of 7- and 19-month-old Tg2576 mice using multimodal in vivo imaging: limitations as a translatable model of Alzheimer's disease

With 90% of neuroscience clinical trials failing to see efficacy, there is a clear need for the development of disease biomarkers that can improve the ability to predict human Alzheimers disease (AD) trial outcomes from animal studies. Several lines of evidence, including genetic susceptibility and disease studies, suggest the utility of fluorodeoxyglucose positron emission tomography (FDG-PET) as a potential biomarker with congruency between humans and animal models. For example, early in AD, patients present with decreased glucose metabolism in the entorhinal cortex and several regions of the brain associated with disease pathology and cognitive decline. While several of the commonly used AD mouse models fail to show all the hallmarks of the disease or the limbic to cortical trajectory, there has not been a systematic evaluation of imaging-derived biomarkers across animal models of AD, contrary to what has been achieved in recent years in the Alzheimers Disease Neuroimaging Initiative (ADNI) (Miller, 2009). If animal AD models were found to mimic endpoints that correlate with the disease onset, progression, and relapse, then the identification of such markers in animal models could afford the field a translational tool to help bridge the preclinical-clinical gap. Using a combination of FDG-PET and functional magnetic resonance imaging (fMRI), we examined the Tg2576 mouse for global and regional measures of brain glucose metabolism at 7 and 19 months of age. In experiment 1 we observed that at younger ages, when some plaque burden and cognitive deficits have been reported, Tg2576 mice showed hypermetabolism as assessed with FDG-PET. This hypermetabolism decreased with age to levels similar to wild type (WT) counterparts such that the 19-month-old transgenic (Tg) mice did not differ from age matched WTs. In experiment 2, using cerebral blood volume (CBV) fMRI, we demonstrated that the hypermetabolism observed in Tg mice at 7 months could not be explained by changes in hemodynamic parameters as no differences were observed when compared with WTs. Taken together, these data identify brain hypermetabolism in Tg2576 mice which cannot be accounted for by changes in vascular compliance. Instead, the hypermetabolism may reflect a neuronal compensatory mechanism. Our data are discussed in the context of disease biomarker identification and target validation, suggesting little or no utility for translational based studies using Tg2576 mice.

Magnetic resonance imaging detection and time course of cerebral microhemorrhages during passive immunotherapy in living amyloid precursor protein transgenic mice.

In recent years immunotherapy-based approaches for treating Alzheimers disease have become the subject of intensive research. However, an important mechanistic-related safety concern is exacerbation of the risk of microhemorrhage that may be associated with fast removal of amyloid-β (Aβ) deposits found in blood vessels or brain parenchyma. Rapid in vivo detection of microhemorrhages in living amyloid precursor protein transgenic mice has not been described, and histological analysis can take several months before this risk is assessed. Aged transgenic mice were divided into two groups that would undergo longitudinal passive immunotherapy for 12 or 18 weeks. 6G1, a nonselective anti-Aβ monoclonal antibody, and 8F5, a more selective antioligomeric Aβ monoclonal antibody, were examined in both longitudinal studies. High-resolution T2*-weighted magnetic resonance microscopy (100 × 100 × 400 μm) was used for microhemorrhage detection in vivo. Cerebral microhemorrhages by magnetic resonance imaging were compared with histological hemosiderin staining in each animal; results showed that T2*-weighted magnetic resonance microscopy can reliably detect microhemorrhages of ≥60 μm in diameter at baseline and after 12 to 18 weeks of treatment in the same animals in vivo. This correlated significantly with histological readings. This new imaging safety biomarker can be readily applied to preclinical antibody screening in a longitudinal manner. 6G1 and 8F5, however, both increased microhemorrhage incidence in aged amyloid precursor protein transgenic mice compared with their baseline and vehicle treatment. A highly selective antibody for soluble Aβ is needed to address the question of whether antibodies that do not bind to deposited Aβ have microhemorrhage liability.

Abeta-oligomer selective antibody A-887755 exhibits a favorable profile for Alzheimer's disease immunotherapy compared to Abeta-peptide unselective antibodies

Background: Although Amyloid-beta (Abeta) fibrils within Abeta plaques are a hallmark of Alzheimer’s disease (AD) during the past decade a paradigm shift occurred accounting Abeta-oligomers as the crucial neuropathogenic culprit of AD. In addition, Abeta-monomer was shown to be produced and metabolized in high amounts and associated with physiological brain function (Bateman et al. 2006). Here, we show that a synthetic, homogenous Abeta20-42 oligomer preparation (named Abeta20-42 Globulomer, Barghorn et al. 2005) is useful to generate monoclonal antibodies that selectively detect Abeta-oligomers in various synthetic and biological Abeta preparations while discriminating Abeta-monomer or Abeta-fibrils. We propose such Abeta-oligomer selective antibodies, like A-887755, as favorable for passive AD immunotherapy. Methods: We tested the capability of the Globulomer-specific antibody A-887755 to prevent Globulomer binding to primary hippocampal neurons in an excess of monomeric Abeta40/42 peptide thus trying to mimic the in vivo situation of passive AD immunotherapy. Results: Under cell culture conditions Abeta20-42 Globulomers bind in a punctuated manner to dendritic processes of primary hippocampal neurons. Preincubation with Globulomerspecific antibody A-887755 at equimolar concentrations prevents Abeta20-42 Globulomer binding even in the presence of excess Abeta40/42 monomer. In contrast, 18-fold higher concentrations of Abeta-peptide unselective antibodies under otherwise identical conditions do not prevent Abeta20-42 Globulomer binding. Conclusions: Abetaoligomers are regarded as the major neuropathogenic Abeta-species in AD. Passive immunotherapy with Abeta-peptide unselective antibodies can in principle neutralize Abeta-oligomer-mediated effects, although these antibodies also recognize Abeta-fibrils and Abeta-monomer. We propose that exclusively targeting Abeta-oligomers with Globulomer selective antibodies like A-887755 has major advantages. Firstly, targeting exclusively Abeta-oligomers as the neuropathogenic culprit of AD minimizes potential side effects by not binding to other Abeta-species such as Abeta-monomer, sAPPalpha, and Abeta-fibrils. Secondly, A-887755 is not consumed by binding to non disease related Abeta-peptide species, in particular Abeta-monomer. In summary these characteristics synergistically provide Globulomer specific antibody A-887755 with a highly favourable profile compared to Abeta-peptide unselective antibodies for AD immunotherapy.

An ELISA-based method for the quantification of incorporated BrdU as a measure of cell proliferation in vivo.

In this study, we describe a new rapid and versatile method to determine the BrdU content of DNA in brain tissues dissected from BrdU-treated rats. Different to already existing BrdU ELISAs the method is suitable for the assessment of BrdU incorporation in ex vivo experiments as it is based on the analysis of tissue extracts instead of immobilized cells. The method comprises the preparation of DNA extracts from dissected tissues, the immobilization of BrdU-containing DNA with an anti-BrdU antibody and quantification of the incorporated BrdU by a peroxidase-conjugated anti-BrdU antibody. Validating the new assay in vitro, we found a clear-cut dependency of the ELISA signal from the time SKNSH neuroblastoma cells had been exposed to BrdU. Parallel studies with existing ELISAs and a parallel immunocytochemical determination of BrdU positive cells revealed comparable results. In vivo experiments showed a virtually linear relationship between the BrdU immunoreactivity in the hippocampus and the time rats have been exposed to BrdU. Repeating the determination of the BrdU content of the same set of tissue samples revealed reproducible relative differences of the ELISA signals. This was true for protocols using purified DNA as well as crude DNA extracts. For the sensitivity and reproducibility of the method heat denaturation of the DNA prior to the analysis in the ELISA was crucial. In rats treated with electroconvulsion the BrdU content of the hippocampus, determined by the new ELISA, was increased to 225% of controls. In a parallel immunohistochemical study, the number of BrdU positive cells was comparably increased to 251% of controls. The assay thus provides a rapid method to detect changes of cell proliferation in dissected brain tissues and other proliferative tissues. With appropriate protocols, the assay may also be used to assess the generation of particular cell types like neurons in neurogenic areas.

Restoration of home cage activity in Tg2576 mice by immunotherapy with the Ab-oligomer selective antibody A-887755

endogenous neurogenesis occurs, we hypothesized that the neurotrophic factors may improve cognition by increasing endogenous neurogenesis. Therefore, to test this hypothesis, we assessed whether stem cell transplants improved cognition by increasing neurogenesis in the brain. Methods: To test this hypothesis, wild-type neural stem cells derived from Tg2576 X B6SJL/F1 mice were stereotactically injected into the brains of Tg2576 mice (15 mo), followed by BrdU injections. After one month, mice behavior was assessed using various tasks (Morris watermaze, novel object, novel odor task). Mice were subsequently sacrificed. We examined the effect of stem cells on pathology and neurogenesis using stereology. Results: Stem cell transplants in Tg2576 mice rescued cognitive deficits observed in these animals at 15 mo. as assessed with various behavioral tasks. The improvement in cognitive deficits was correlated with neurogenesis in the brain. Conclusions: Here we show that stem cell transplants in AD transgenic mice rescue the cognitive deficits and that this is associated with an increase in neurogenesis.

P2-483: Biophysical characterization of soluble amyloid-β peptide oligomers

to the inhibitor structure, as well as filling extra binding pockets with additional functional groups. The optimised lead was shown to have enhanced activity with an IC50 towards BACE1 of 37 M. Analogues with IC50 100 M towards BACE1 were tested in the cellular assay, and selected compounds have shown promising cellular activity with EC50 10 M. Conclusions: The rational design approach using de novo design software SPROUT has successfully delivered a new cellular active BACE1 inhibitor scaffold.