Work with the Alzheimer’s disease-related synthetic peptide beta-amyloid (Aβ) is a challenging task because of its disadvantageous dissolution properties and high propensity for aggregation. Recently, a new synthetic derivative, iso-Aβ 42 , has been introduced, which is a precursor of Aβ 42 , and it offers advantages as concerns its synthesis and use for sample preparation. These two Aβ forms showed high similarity in their biological effects, as well as in their main structural characteristics under well-chosen experimental circumstances. When we changed these conditions, considerable dissimilarities appeared in the aggregation properties of the two peptides. In the present study, the aggregation pathways of native and precursor-derived Aβ 42 oligomers were compared in a physiological buffer with and without divalent metal ions (Ca 2+ /Mg 2+ ). The presence of these ions influenced the Aβ conformations, the morphology as well as formation dynamics of aggregates in a different manner, as it was demonstrated by thioflavin-T-binding experiments, transmission electron microscopy and electronic circular dichroism measurements. Namely, the aggregation of native Aβ 42 to fibrils was facilitated, while the aggregation of precursor-derived Aβ 42 was hindered by these divalent metal ions. The observed differences in the aggregation had an impact also on the biological efficiency of native and precursor-derived Aβ 42 as it was elucidated by viability assays with enhanced sensitivity on primary endothelial cell cultures. Using replica exchange molecular dynamics, we modeled the conformational ensembles of the two investigated Aβ variants evolving during preparation process. We found considerable differences in the probability distribution of the conformers that can explain the observed dissimilarities in their aggregation properties.

IV. A novel and simple fluorescence method for the measurement of presynaptic vesicular zinc release in acute hippocampal slices with a fluorescence plate reader. II. Membrane-lipid therapy in operation: the Hsp co-inducer BGP-15 activates stress signal transduction pathways by remodeling plasma membrane rafts. III. Hydroximic acid derivatives: pleiotrophic Hsp co-inducers restoring homeostasis and robustness. Abbreviations αSN Alpha-synuclein protein Aβ Amyloid-beta peptide ACSF Artificial cerebrospinal fluid AD Alzheimer " s disease ADDLs Aβ-derived diffusible ligands APP Amyloid precursor protein ASPD Native-amylospheroids BCA Bicinchoninic acid CDK5 Cyclin-dependent kinase 5 DLBD Diffuse Lewy body disease DMSO Dimethyl-sulfoxide DNP 2,4-dinitrophenol FBS Fetal bovine serum fEPSP Field excitatory postsynaptic potentials GSK-3β Glycogen synthase kinase 3 beta HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid LBD Lewy body diseases LBV Lewy body variant of Alzheimer " s disease LDH Lactate dehydrogenase LTP Long-term potentiation MAP Microtubule associated protein MEM Minimal essential medium NAC Non-amyloid component of Alzheimer " s disease amyloid NACP NAC precursor = alpha-synuclein NFTs Neurofibrillary tangles NMDA N-methyl-D-aspartate NMDAR N-methyl-D-aspartate receptor NR2B Subunit of NMDAR PBS Phosphate buffered saline PD Parkinson " s disease PI Propidium iodide PP-1 Protein phosphatase 1 PP-2A Protein phosphatase 2A RT Room temperature SDS-PAGE Sodium dodecyl sulphate-polyacrylamide gel electrophoresis SEM Standard error of mean TBS Theta-burst stimulation TEM Transmission electron microscopy upH 2 O MilliQ ultrapure water ZEN Zinc-enriched neuron ZnT Zinc transporter Alzheimer " s disease (AD) is a heterogeneous, progressive neurodegenerative disorder representing the most common cause of cognitive failure and dementia in older human patients. Extracellular deposition of beta amyloid peptide, intracellular formation of neurofibrillar tangles (NFT) caused by hyperphosphorylated tau protein and oxidative stress induced by impaired metabolic pathways and metals are the hallmarks of the disease primary theory for the cause of AD is the overproduction and/or impaired clearance of amyloid-beta (Aβ) peptides derived from amyloid precursor protein (APP), especially the The secondary hypothesis is the " metal hypothesis " which proposes the interaction of Aβ with specific metal ions that could enhance Aβ aggregation, pathogenicity and finally cause Brain tissues of AD patients after autopsy contain two main characteristic lesions: extracellular amyloid plaques and intracellular neurofibrillary tangles which are formed by hyperphosphorylated tau protein (Goedert and Crowther, 1989, Selkoe, 2003). There are two main forms of amyloid depositions: senile (or neuritic) and diffuse plaques. Senile plaques (originally observed by Alois Alzheimer) are extracellular spherical structures around 50–200 µm in diameter which contain a central amyloid core enriched …

Background and Aims Unnatural self-organizing biomimetic polymers (foldamers) emerged as promising materials for biomolecule recognition and inhibition. Our goal was to construct multivalent foldamer-dendrimer conjugates which wrap the synaptotoxic β-amyloid (Aβ) oligomers with high affinity through their helical foldamer tentacles. Oligomeric Aβ species play pivotal role in Alzheimer's disease, therefore recognition and direct inhibition of this undruggable target is a great current challenge. Methods and Results Short helical β-peptide foldamers with designed secondary structures and side chain chemistry patterns were applied as potential recognition segments and their binding to the target was tested with NMR methods (saturation transfer difference and transferred-nuclear Overhauser effect). Helices exhibiting binding in the µM region were coupled to a tetravalent G0-PAMAM dendrimer. In vitro biophysical (isothermal titration calorimetry, dynamic light scattering, transmission electron microscopy and size-exclusion chromatography) and biochemical tests (ELISA and dot blot) indicated the tight binding between the foldamer conjugates and the Aβ oligomers. Moreover, a selective low nM interaction with the low molecular weight fraction of the Aβ oligomers was found. Ex vivo electrophysiological experiments revealed that the new material rescues the long-term potentiation from the toxic Aβ oligomers in mouse hippocampal slices at submicromolar concentration. Conclusions The combination of the foldamer methodology, the fragment-based approach and the multivalent design offers a pathway to unnatural protein mimetics that are capable of specific molecular recognition, and has already resulted in an inhibitor for an extremely difficult target.

Uj, standardizalhato modszert dolgoztunk ki toxikus �beta-amiloid (Abeta) 1-42 peptid oligomerek előallitasara, a preparalt oligomereket fiziko-kemiai modszerekkel jellemeztuk. Ket uj neuroprotektiv peptidmimetikum vegyuletcsaladot talaltunk, ezek az anyagok megvedik a neuronokat az Alzheimer-kor (AK) allatmodelljeben az Abeta neurotoxikus hatasatol. Mindket vegyuletcsoportot szabadalmilag vedjuk, mint az AK potencialis gyogyszerjelolt vegyuleteit. Uj ex vivo modszert dolgoztunk ki az Abeta peptidek toxicitasanak meresere (patkany hippocampus szelet, MTT-teszt), a modszer alkalmas az uj neuroprotektiv vegyuleteink aktivitasmeresere is. Az ex vivo hippocampus szeleteket sikerrel alkalmaztuk a neuronalis plaszticitas (LTP) meresere, az Abeta-toxicitas meghatarozasara, multielektrod array (MEA) technikaval. In vivo, egysejt-elvezeteses elektrofiziologiai meresekkel bizonyitottuk az uj peptidmimetikumaink neuroprotektiv hatasat. Proteomikai modszerekkel azonositottuk az Abeta peptidekkel kolcsonhatasba lepő feherjeket, ezek elsősorban plazmamembran, ill. intraneuronalis feherjek (mitokondrium, endoplazmas reticulum, mikrotubularis rendszer). Az intraneuronalis feherjek es az Abeta peptidek kolcsonhatasai kulcsszerepet jatszhatnak az AK patogeneziseben. Igazoltuk, hogy a Zn2+ ionok toxikus Abeta-aggregatumok kepződeset indukaljak. Az AK transzgen allatmodelljen bizonyitottuk, hogy a Zn-kelatorok (pl. Perindopril) neuroprotektiv hatasuak. Uj AK-allatmodellt dolgoztunk ki az Abeta oligomerek icv bevitelevel. | A new method was introduced for the preparation of toxic beta-amyloid (Abeta) 1-42 oligomers, these assemblies were characterized with physicochemical methods. Two families of novel neuroprotective peptidomimetics were found, these substances protect neurons against the toxic effect of Abeta in tg mouse models of Alzheimer’s disease (AD). Both groups of the novel substances will be patented as putative drug candidates for AD treatment. A new ex vivo method was introduced for toxicity measurement of Abeta peptides (rat hippocampal slices, MTT-assay); this method proved to be suitable for activity measurement of the novel neuroprotective substances. Hippocampal slices were successfully used for measurement of neuronal plasticity (LTP) for demonstrating neurotoxicity of Abeta aggregates, applying multielectrode array (MEA) technique. The neuroprotective effect of our novel peptidomimetics was demonstrated also in vivo, using one-cell electrophysiology. Proteomic methods were used for identification of proteins interacting with Abeta peptides; these are mainly plasma membrane and intraneuronal (mitochondrial, endoplasmatic reticular and microtubular) proteins. Interaction of intracellular proteins with Abeta may play key role in AD pathogenesis. The role of Zn2+ ions in formation of toxic Abeta-aggregates was demonstrated. Zn2+-chelators (e.g. Perindopril) were neuroprotective in a tg-mouse model of AD. A new AD rat model was introduced using icv administration of synthetic Abeta oligomers.

Oligomeric amyloid-β is currently of interest in amyloid-β mediated toxicity and the pathogenesis of Alzheimer's disease. Mapping the amyloid-β interaction partners could help to discover novel pathways in disease pathogenesis. To discover the amyloid-β interaction partners, we applied a protein array with more than 8100 unique recombinantly expressed human proteins. We identified 324 proteins as potential interactors of oligomeric amyloid-β. The Gene Ontology functional analysis of these proteins showed that oligomeric amyloid-β bound to multiple proteins with diverse functions both from extra and intracellular localizations. This undiscriminating binding phenotype indicates that multiple protein interactions mediate the toxicity of the oligomeric amyloid-β. The most highly impacted cellular system was the protein translation machinery. Oligomeric amyloid-β could bind to altogether 24 proteins involved in translation initiation and elongation. The binding of amyloid-β to purified rat hippocampal ribosomes validated the protein array results. More importantly, in vitro translation assays showed that the oligomeric amyloid-β had a concentration dependent inhibitory activity on translation. Our results indicate that the inhibited protein synthesis is one of the pathways that can be involved in the amyloid-beta induced neurotoxicity.

The disordered tubulin polymerization promoting protein (TPPP/p25) was found to be co-enriched in neuronal and glial inclusions with α-synuclein in Parkinson disease and multiple system atrophy, respectively; however, co-occurrence of α-synuclein with β-amyloid (Aβ) in human brain inclusions has been recently reported, suggesting the existence of mixed type pathologies that could result in obstacles in the correct diagnosis and treatment. Here we identified TPPP/p25 as an interacting partner of the soluble Aβ oligomers as major risk factors for Alzheimer disease using ProtoArray human protein microarray. The interactions of oligomeric Aβ with proteins involved in the etiology of neurological disorders were characterized by ELISA, surface plasmon resonance, pelleting experiments, and tubulin polymerization assay. We showed that the Aβ(42) tightly bound to TPPP/p25 (K(d) = 85 nm) and caused aberrant protein aggregation by inhibiting the physiologically relevant TPPP/p25-derived microtubule assembly. The pair-wise interactions of Aβ(42), α-synuclein, and tubulin were found to be relatively weak; however, these three components formed soluble ternary complex exclusively in the absence of TPPP/p25. The aggregation-facilitating activity of TPPP/p25 and its interaction with Aβ was monitored by electron microscopy with purified proteins by pelleting experiments with cell-free extracts as well as by confocal microscopy with CHO cells expressing TPPP/p25 or amyloid. The finding that the interaction of TPPP/p25 with Aβ can produce pathological-like aggregates is tightly coupled with unusual pathology of the Alzheimer disease revealed previously; that is, partial co-localization of Aβ and TPPP/p25 in the case of diffuse Lewy body disease with Alzheimer disease.

Two pyridine derivatives, DMAP and ENDIP, have been investigated as possible metal chelators in the therapy of Alzheimer's disease.Their complex formation with Cu(II) and Zn(II) were characterised in detail.In the case of ENDIP a high stability tetradentate ML complex is formed at physiological pH both with Cu(II) and Zn(II).DMAP was found to be a weaker metal binder.At physiological pH, it forms a bidentate ML complex with Zn(II) and MLH -1 and ML 2 complexes with Cu(II), depending on the metal ion to ligand ratio.Fluorescence spectroscopy and dynamic light scattering measurements proved that ENDIP effectively competes with aggregated amyloid-b peptides (Ab) for both Cu(II) and Zn(II) and thus is able to prevent the metal ion-induced amyloid aggregation and to resolubilise amyloid precipitates.

β-Amyloid (Aβ) peptides play a crucial role in the pathology of the neurodegeneration in Alzheimer's disease (AD). Biological experiments (both in vitro and animal model studies of AD) require synthetic Aβ peptides of standard quality, aggregation grade, neurotoxicity and water solubility. The synthesis of Aβ peptides has been difficult, owing to their hydrophobic character, poor solubility and high tendency for aggregation. Recently an isopeptide precursor (iso-Aβ(1–42)) was synthesized by Fmoc-chemistry and transformed at neutral pH to Aβ(1–42) by O→N acyl migration in a short period of time. We prepared the same precursor peptide using Boc-chemistry and studied the transformation to Aβ(1–42) by acyl migration. The peptide conformation and aggregation processes were studied by several methods (circular dichroism, atomic force and transmission electron microscopy, dynamic light scattering). The biological activity of the synthetic Aβ(1–42) was measured by ex vivo (long-term potentiation studies in rat hippocampal slices) and in vivo experiments (spatial learning of rats). It was proven that O→N acyl migration of the precursor isopeptide results in a water soluble oligomeric mixture of neurotoxic Aβ(1–42). These oligomers are formed in situ just before the biological experiments and their aggregation grade could be standardized.

Two pyridine derivatives, DMAP and ENDIP, have been investigated as possible metal chelators in the therapy of Alzheimer's disease. Their complex formation with Cu(ii) and Zn(ii) were characterised in detail. In the case of ENDIP a high stability tetradentate ML complex is formed at physiological pH both with Cu(ii) and Zn(ii). DMAP was found to be a weaker metal binder. At physiological pH, it forms a bidentate ML complex with Zn(ii) and MLH(-1) and ML(2) complexes with Cu(ii), depending on the metal ion to ligand ratio. Fluorescence spectroscopy and dynamic light scattering measurements proved that ENDIP effectively competes with aggregated amyloid-beta peptides (Abeta) for both Cu(ii) and Zn(ii) and thus is able to prevent the metal ion-induced amyloid aggregation and to resolubilise amyloid precipitates.