Heinz Hillen

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.

Molecular characterization of human and bovine endothelin converting enzyme (ECE‐1)

A membrane‐bound protease activity that specifically converts Big endothelin‐1 has been purified from bovine endothelial cells (FBHE) The enzyme was cleaved with trypsin and the peptide sequencing analysis confirmed it to be a zinc chelating metalloprotease containing the typical HEXXH (HELTH) motif. RT‐PCR and cDNA screens were employed to isolate the complete cDNAs of the bovine and human enzymes. This human metalloprotease was expressed heterologously in cell culture oocytes. The catalytic activity of the recombinant enzyme is the same as that determined for the natural enzyme. The data suggest that the characterized enzyme represents the functional human endothelin converting enzyme ECE‐1.

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.

Aβ-globulomers are formed independently of the fibril pathway

Soluble A beta-oligomers are currently discussed as the major causative species for the development of Alzheimers disease (AD). Consequently, the beta-amyloid cascade hypothesis was extended by A beta-oligomers and their central neuropathogenic role in AD. However, the molecular structure of A beta-oligomers and their relation to amyloid fibril formation remains elusive. Previously we demonstrated that incubation of A beta(1-42) with SDS or fatty acids induces the formation of a homogeneous globular A beta-oligomer termed A beta-globulomer. In this study we investigated the role of A beta-globulomers in the aggregation pathway of A beta-peptide. We used in vitro assays such as thioflavin-T binding and aggregation inhibitors like Congo red to reveal that A beta-peptide in its A beta-globulomer conformation is a structural entity which is independent from amyloid fibril formation. In addition, cellular Alzheimers-like plaque forming assays show the resistance of A beta-globulomers to deposition as amyloid plaques. We hypothesize that a conformational switch of A beta is decisive for either fibril formation or alternatively and independently A beta-globulomer formation.

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.

Monomeric Structures of the Zymogen and Active Catalytic Domain of Complement Protease C1r: Further Insights into the C1 Activation Mechanism

C1r is the serine protease (SP) that mediates autoactivation of C1, the complex that triggers the classical complement pathway. We have determined the crystal structure of two fragments from the human C1r catalytic domain, each encompassing the second complement control protein (CCP2) module and the SP domain. The wild-type species has an active structure, whereas the S637A mutant is a zymogen. The structures reveal a restricted hinge flexibility of the CCP2-SP interface, and both are characterized by the unique alpha-helical conformation of loop E. The zymogen activation domain exhibits high mobility, and the active structure shows a restricted access to most substrate binding subsites. Further implications relevant to the C1r self-activation process are derived from protein-protein interactions in the crystals.

Serine protease inhibitor nafamostat given before reperfusion reduces inflammatory myocardial injury by complement and neutrophil inhibition.

Animal data strongly support a role for inflammation in myocardial ischemia reperfusion injury. Attempts at cardioprotection by immunomodulation (such as with the specific C5 antibody pexelizumab) in humans have been disappointing. We hypothesized that a broader spectrum antiinflammatory agent might yield successful cardioprotection. The serine protease inhibitor nafamostat (FUT-175), which is already in clinical use, is a potent antiinflammatory synthetic serine protease inhibitor with anticomplement activity that we tested in a well-established rabbit model of 1 hour of myocardial ischemia followed by 3 hours of reperfusion. Compared to vehicle-treated animals, nafamostat (1 mg/kg of body weight) administered 5 minutes before reperfusion significantly reduced myocardial injury assessed by plasma creatine kinase activity (38.1 ± 6.0 versus 57.9 ± 3.7I U/g protein; P < 0.05) and myocardial necrosis (23.6 ± 3.1% versus 35.7 ± 1.0%; P < 0.05) as well as myocardial leukocyte accumulation (P < 0.05). In parallel in vitro studies, Nafamostat was a significantly more potent broad spectrum complement suppressor than C1 inhibitor. Nafamostat appears to have capability as an inhibitor of both complement pathways and as a broad-spectrum antiinflammatory agent by virtue of its serine protease inhibition. Administration of nafamostat before myocardial reperfusion after ischemia produced significant, dose-dependant cardioprotection. Reduced leukocyte accumulation and complement activity seem involved in the mechanism of this cardioprotective effect.

Monoclonal antibodies against human endothelin-converting enzyme-1.

Endothelin-converting enzyme-1 (ECE-1) is a membrane-bound metalloprotease which specifically converts the inactive precursor big-endothelin-1 (big ET-1) to the vasoactive endothelin-1 (ET-1). Six different mouse hybridoma cell lines have been generated secreting monoclonal antibodies specific to human ECE-1. These antibodies have been proven useful in a fast and efficient one-step purification of membrane-bound ECE-1 as well as of artificial soluble ECE-1 by immunoaffinity chromatography. The antibodies are suitable for a quantification of ECE-1 in solution by a sandwich-ELISA and for the immunohistochemical detection of ECE-1 in the cell membrane.

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.

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.