Ingrid Van der Auwera

Liposomal vaccines with conformation-specific amyloid peptide antigens define immune response and efficacy in APP transgenic mice

We investigated the therapeutic effects of two different versions of Aβ1–15 (16) liposome-based vaccines. Inoculation of APP-V717IxPS-1 (APPxPS-1) double-transgenic mice with tetra-palmitoylated amyloid 1–15 peptide (palmAβ1–15), or with amyloid 1–16 peptide (PEG-Aβ1–16) linked to a polyethyleneglycol spacer at each end, and embedded within a liposome membrane, elicited fast immune responses with identical binding epitopes. PalmAβ1–15 liposomal vaccine elicited an immune response that restored the memory defect of the mice, whereas that of PEG-Aβ1–16 had no such effect. Immunoglobulins that were generated were predominantly of the IgG class with palmAβ1–15, whereas those elicited by PEG-Aβ1–16 were primarily of the IgM class. The IgG subclasses of the antibodies generated by both vaccines were mostly IgG2b indicating noninflammatory Th2 isotype. CD and NMR revealed predominantly β-sheet conformation of palmAβ1–15 and random coil of PEG-Aβ1–16. We conclude that the association with liposomes induced a variation of the immunogenic structures and thereby different immunogenicities. This finding supports the hypothesis that Alzheimers disease is a “conformational” disease, implying that antibodies against amyloid sequences in the β-sheet conformation are preferred as potential therapeutic agents.

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.

Virosome-based active immunization targets soluble amyloid species rather than plaques in a transgenic mouse model of Alzheimer’s disease

Active vaccination with amyloid peptides shows promise for the treatment and prevention of Alzheimer’s disease (AD). Several studies in transgenic mouse models of AD have revealed the potency of vaccination to prevent or even clear amyloid plaques from mouse brain. However, the idea that soluble oligomeric species of β-amyloid (Aβ), rather than plaques, trigger the disease has gained momentum, and current active vaccination strategies affect the levels of total or soluble brain Aβ little or not at all. We describe an active vaccination method based on Aβ1-16 presented on the surface of virosomes, which triggered a dramatic decrease in both soluble Aβ40 (75% reduction; p=0.01) and soluble Aβ42 (62% reduction; p=0.03) in a double transgenic mouse model of AD. Whereas Aβ40 and Aβ42 levels in the insoluble fraction tended to be reduced (by 30% and 27%, respectively), the number of thioflavine-S-positive amyloid plaques was not affected. The high specific antibody responses, obtained without eliciting T-cell reactivity, demonstrate that immunostimulating reconstituted influenza virosomes are a promising antigen carrier system against the neuropathology of AD.

Preparation of Cyclo-Phen-Type Ligands: Chelators of Metal Ions as Potential Therapeutic Agents in the Treatment of Neurodegenerative Diseases

Many recent studies have shown the major role played by metal ions (copper, zinc, iron, ...) in the modification of the folding and/or the aggregation of proteins that leads to serious pathologies. Several neurodegenerative diseases (Alzheimer’s disease, spongiform encephalopathies, Parkinson’s disease, Huntington’s disease, ...) involve similar disastrous interactions between metal ions and proteins. In the case of Alzheimer’s disease, the pathology is associated with the aggregation of b-amyloid peptides (Ab) in the brain, which leads to the formation of amyloid plaques. The accumulation of redox-active metal ions in these amyloid plaques is probably responsible for the oxidative stress which induces neuronal lesions in the brain that result in irreversible loss of intellectual faculties. The use of a metal ligand like Clioquinol led to improvements in patients suffering from Alzheimer’s disease and indicated that therapeutic approaches are possible with metal ion chelators in neurodegenerative diseases. Among Cu/Zn chelators capable of solubilizing Ab from post mortem brain tissue, interesting results were obtained with bathophenanthroline and bathocuproine, two ligands based on the 1,10phenanthroline structure. However, due to the presence of sulfate residues, these chelators are too hydrophilic to cross the blood brain barrier; consequently their use in vivo cannot be envisaged.

Application of the Fertilase-laser system versus the conventional mechanical method to immobilize spermatozoa for intracytoplasmic sperm injection. A randomized controlled trial.

A randomized controlled trial was set up to test the hypothesis that the fertilization rate of oocytes after intracytoplasmic sperm injection (ICSI) is higher after immobilization of the spermatozoa with the Fertilase®-laser system technology than after immobilization of the spermatozoa with the conventional mechanical method. Metaphase II oocytes were injected with spermatozoa that were immobilized with the conventional mechanical method (group A, n = 177) or with spermatozoa that were immobilized with the Fertilase-laser system technology (group B, n = 179). The fertilization rate per successfully injected oocyte was comparable in group A (62.6%; 92/147) and in group B (56.3%; 89/158)(p = 0.3). No difference could be observed in fertilization rates of oocytes injected with spermatozoa that were immobilized with the Fertilase-laser system technology compared to spermatozoa immobilized with the conventional mechanical method.

P4-245: Nanobodies™ targeting amyloid beta as potential therapeutics for Alzheimer’s disease

discovery of a GSI possessing minimal inhibition of Notch processing and maximal inhibition of APP processing to reduce 40/42 levels. Utilizing HTS screening of the Wyeth and ArQule compound collections, we have identified the lead, 4-chloro-N-[(1S,2S)-1-(hydroxymethyl)-2-methylbutyl]-benzenesulfonamide, which not only inhibited -secretase cleavage of APP (EC50 40 and 42 2078 nM and 1938 nM, respectively) but also was sparing of Notch cleavage (EC50 20,000 nM). Structure-activity relationships in this new series and the discovery of more potent GSIs will be discussed.

O3-05-03: Therapeutic effects of passive immunization with an Aβ-oligomer– specific antibody in APP transgenic mice

pounds that can prevent amyloid oligomer/fibril formation. The targeted amyloids include amyloid-beta of Alzheimer’s, prion, alpha-synuclein of Parkinson’s, transthyretin, insulin, and Ig light chains. Screening techniques for assessing the effectiveness of small molecule inhibitors include a variety of physical-chemical and toxicity assays (e.g., turbidity and Congo red or thioflavin binding), morphological imaging (EM; AFM), spectroscopy (CD; NMR), and cytotoxicity and biochemistry (cell cultures; protease-resistance; immunoblotting). Methods: X-ray fiber diffraction from amyloids shows that their two key structural features are the 4.7 Å-distance between H-bonded beta-strands and the side chain-dependent 5-15 Å-distance between beta-pleated sheets. The widths of these reflections are related reciprocally to the extent of H-bonding and intersheet stacking. Thus, by measuring the integral widths and integrated intensities of these signature reflections, we used -ray fiber diffraction to test the inhibitory effect of a number of small aromatic molecules and N-methylated Abeta16-22 on the aggregation of different Abeta analogues (sequences 1-40, 11-25, 12-28, 17-28, and 16-22). Results: X-ray diffraction assessment showed that Cat’s claw, tannic acid, and quinine were more effective than curcumin, morin, and melatonin in impeding amyloid fiber formation, based on the criterion of crystallite volume. As their concentration increased, tetracycline and cotinine decreased the coherent length of the 4.7 Å-reflection, and Cat’s claw abolished this reflection. For the N-methylated and control peptides, there were no substantial differences in spacings and coherent lengths; however, the relative volumes of the beta-crystallites, which was calculated from the magnitude of the intensities, decreased with increase in the concentration of the methylated peptide, which may be accounted for by the its binding to the monomer or preamyloid oligomer of the non-methylated peptide. Conclusions: Fiber diffraction can help specify in an in vitro screen whether an amyloidophilic compound inhibits fibril formation, and if so, whether by impeding H-bonding or by altering intersheet interactions. Developing and implementing a high-throughput screen should be straightforward using robotic sample preparation, sample movement through a high-intensity -ray beam, automated data collection with fast electronic detectors, and automated analysis of the -ray data.