Viktor Szegedi

Divergent effects of Aβ1–42 on ionotropic glutamate receptor-mediated responses in CA1 neurons in vivo

Aggregated Abeta1-42 is hypothesized to be the central cause of Alzheimers disease. However, early changes in synaptic activity may be detected in the disease long before a significant cell loss is manifested. Despite the fact that Abeta1-42 interference with long-term potentiation (LTP) and the field excitatory postsynaptic potential (fEPSP) is well documented, the exact mechanism of these events remains to be clarified. Here we studied the effects of iontophoretically applied Abeta1-42 on the neuronal firing evoked in vivo on the CA1 hippocampal neurons of Wistar rats by different agonists of the ionotropic glutamate receptors: N-methyl-d-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and kainic acid (KA). NMDA elicited firing enhanced in all of the measured cells; in contrast, the AMPA-mediated responses decreased significantly after Abeta1-42 ejection. The changes in KA-evoked responses to Abeta1-42 revealed two types of cells. In the first type, the KA-mediated firing remained at the control level, while in the second type, Abeta1-42 attenuated the KA-evoked responses. A protective pentapeptide, Leu-Pro-Tyr-Phe-Asp-amide, was used to verify the specificity of these beta-amyloid-elicited effects. The pentapeptide protected against the modulatory effects of Abeta1-42 on the NMDA and AMPA responses. In conclusion, we have shown that Abeta1-42 exerts divergent effects on the activity of the ionotropic glutamate receptors in vivo. These results suggest that the LTP disruption and fEPSP attenuation seen after Abeta1-42 application are in part due to the altered function of these receptors.

Controlled in situ preparation of Aβ(1–42) oligomers from the isopeptide “iso-Aβ(1–42)”, physicochemical and biological characterization

Beta-amyloid (A beta) peptides play a crucial role in the pathology of the neurodegeneration in Alzheimers disease (AD). Biological experiments (both in vitro and animal model studies of AD) require synthetic A beta peptides of standard quality, aggregation grade, neurotoxicity and water solubility. The synthesis of A beta peptides has been difficult, owing to their hydrophobic character, poor solubility and high tendency for aggregation. Recently an isopeptide precursor (iso-A beta(1-42)) was synthesized by Fmoc-chemistry and transformed at neutral pH to A beta(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 A beta(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 A beta(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 A beta(1-42). These oligomers are formed in situ just before the biological experiments and their aggregation grade could be standardized.

A Foldamer-Dendrimer Conjugate Neutralizes Synaptotoxic β-Amyloid Oligomers

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 Alzheimers 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.

Endomorphin-2, an endogenous tetrapeptide, protects against Aβ1–42 in vitro and in vivo

The underlying cause of Alzheimers disease (AD) is thought to be the β‐amyloid aggregates formed mainly by Aβ1–42 peptide. Protective pentapeptides [e.g., Leu‐Pro‐Phe‐Phe‐Asp (LPFFD)] have been shown to prevent neuronal toxicity of Aβ1–42 by arresting and reversing fibril formation. Here we report that an endogenous tetrapeptide, endomorphin‐2 (End‐2, amino acid sequence: YPFF), defends against Aβ 1–42 induced neuromodulatory effects at the cellular level. Although End‐2 does not interfere with the kinetics of Aβ fibrillogenesis according to transmission electron microscopic studies and quasielastic light scattering measurements, it binds to Aβ1–42 during aggregation, as revealed by tritium‐labeled End‐2 binding assay and circular dichroism measurements. The tetrapeptide attenuates the inhibitory effect on cellular redox activity of Aβ1–42 in a dose‐dependent manner, as measured by 3‐(4,5‐dimethylthiazolyl‐2)‐2,‐5‐diphenyltetrazolium bromide (MIT) assay. In vitro and in vivo electrophysiological experiments show that End‐2 also protects against the field excitatory postsynaptic potential attenuating and the NMDA‐evoked responseenhancing effect of Aβ1–42. Studies using [d‐Ala (2), N‐Me‐Phe (4), Gly (5)‐ol]‐enkephalin (DAMGO), a µ‐opioid receptor agonist, show that the protective effects of the tetrapeptide are not µ‐receptor modulated. The endogenous tetrapeptide End‐2 mayserve as a lead compound for the drug development in the treatment of AD.—Szegedi, V., Juhász, G., Rózsa, E., Juhász‐Vedres, G., Datki, Z., Fülöp, L., Bozsó, Z., Lakatos, A., Laczkó, I., Farkas, T., Kis, Z., Tóth, G., Soós, K., Zarándi, M., Budai, D., Toldi, J., Penke, B. Endomorphin‐2, an endogenous tetrapeptide, protects against Aβ1–42 in vitro and in vivo. FASEB J. 20, E324–E333 (2006)

Tianeptine potentiates AMPA receptors by activating CaMKII and PKA via the p38, p42/44 MAPK and JNK pathways

Impairments of cellular plasticity appear to underlie the pathophysiology of major depression. Recently, elevated levels of phosphorylated AMPA receptor were implicated in the antidepressant effect of various drugs. Here, we investigated the effects of an antidepressant, Tianeptine, on synaptic function and GluA1 phosphorylation using murine hippocampal slices and in vivo single-unit recordings. Tianeptine, but not imipramine, increased AMPA receptor-mediated neuronal responses both in vitro and in vivo, in a staurosporine-sensitive manner. Paired-pulse ratio was unaltered by Tianeptine, suggesting a postsynaptic site of action. Tianeptine, 10 μM, enhanced the GluA1-dependent initial phase of LTP, whereas 100 μM impaired the latter phases, indicating a critical role of GluA1 subunit phosphorylation in the excitation. Tianeptine rapidly increased the phosphorylation level of Ser(831)-GluA1 and Ser(845)-GluA1. Using H-89 and KN-93, we show that the activation of both PKA and CaMKII is critical in the effect of Tianeptine on AMPA responses. Moreover, the phosphorylation states of Ser(217/221)-MEK and Thr(183)/Tyr(185)-p42MAPK were increased by Tianeptine and specific kinase blockers of the MAPK pathways (PD 98095, SB 203580 and SP600125) prevented the effects of Tianeptine. Overall these data suggest that Tianeptine potentiates several signaling cascades associated with synaptic plasticity and provide further evidence that a major mechanism of action for Tianeptine is to act as an enhancer of glutamate neurotransmission via AMPA receptors.

Protein Array Based Interactome Analysis of Amyloid-β Indicates an Inhibition of Protein Translation

Oligomeric amyloid-β is currently of interest in amyloid-β mediated toxicity and the pathogenesis of Alzheimers 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.

An intraperitoneally administered pentapeptide protects against Aβ1-42 induced neuronal excitation in vivo

The underlying cause of Alzheimers disease (AD) is thought to be the accumulation and aggregation of a misfolded protein, amyloid-beta (Abeta). A promising strategy against AD is the application of protective, peptide-based neuroprotective agents that selectively bind to Abeta. We recently described a pentapeptide, LPYFDa, which recognizes Abeta (1-42) and protects neurons against the toxic effects of aggregated Abeta (1-42) both in vitro and in vivo. Our previous work indicated that the in vivo ejection of fibrillar Abeta (1-42) into the hippocampal CA1 region resulted in a massive increase in the NMDA-evoked neuronal firing rate. Our current aim was to study whether intraperitoneally administered LPYFDa is capable of protecting against the synaptotoxic action of fibrillar Abeta (1-42) administered by iontophoresis. Our investigations of the in vivo biodistribution of tritium-labelled LPYFDa and single-unit electrophysiology revealed that LPYFDa readily crosses the blood-brain barrier, and protects the synapses against the excitatory action of fibrillar Abeta (1-42) in a relatively wide temporal window in rat. This pentapeptide may serve as a lead compound for the design of novel drug candidates for the prevention of AD.

Fibrillar Aβ1-42 Enhances NMDA Receptor Sensitivity via the Integrin Signaling Pathway

The aggregated form of amyloid-beta (Abeta) (1-42) has been shown to increase N-methyl-D-aspartic acid (NMDA) evoked neuronal activity in vivo. Here we further characterized this phenomenon by investigating the role of integrin activation and downstream Src kinase activity using in vivo electrophysiology and in vitro intracellular Ca (2+) measurements. Pretreatment of differentiated SH-SY5Y cells with fibrillar Abeta (1-42) markedly enhanced the intracellular calcium increases caused by NMDA receptor (NMDA-R) stimulation. Function blocking antibody against beta1 integrin depressed the facilitatory effects of Abeta (1-42). Similarly, Abeta (1-42) facilitated NMDA-R driven firing of hippocampal neurons in vivo, and this effect was reduced by neutralizing antibody against beta1 integrins. The positive action of Abeta (1-42) on NMDA-R dependent responses was also depressed by an inhibitor known to block Src kinase. These results support the hypothesis that aggregated Abeta (1-42) is recognized by the beta1 subunit containing integrins and may induce a Src kinase dependent NMDA receptor phosphorylation.

Differences between normal and alpha-synuclein overexpressing SH-SY5Y neuroblastoma cells after Aβ(1-42) and NAC treatment

Alpha-synuclein (alphaSN) plays a major role in numerous neurodegenerative disorders, such as Alzheimers disease and Parkinsons disease. Intracellular inclusions containing aggregated alphaSN have been reported in Alzheimers and Parkinsons affected brains. Moreover, a proteolytic fragment of alphaSN, the so-called non-amyloid component of Alzheimers disease amyloid (NAC) was found to be an integral part of Alzheimers dementia related plaques. Despite the extensive research on this topic, the exact toxic mechanism of alphaSN remains elusive. We have taken the advantage of an alphaSN overexpressing SH-SY5Y cell line and investigated the effects of classical apoptotic factors (e.g. H(2)O(2), amphotericin B and ruthenium red) and aggregated disease-related peptides on cell viability compared to wild type neuroblastoma cells. It was found that alphaSN overexpressing cells are more sensitive to aggregated peptides treatment than normal expressing counterparts. In contrast, cells containing elevated amount of alphaSN were less vulnerable to classical apoptotic stressors than wild type cells. In addition, alphaSN overexpression is accompanied by altered phenotype, attenuated proliferation kinetics, increased neurite arborisation and decreased cell motility. Based on these results, the alphaSN overexpressing cell lines may represent a good and effective in vitro model for Alzheimers and Parkinsons disease.

Integrin activation modulates NMDA and AMPA receptor function of CA1 cells in a dose-related fashion in vivo

The large family of heterodimeric, transmembrane cell adhesion receptors, integrins mediate numerous functions in the immature and adult CNS. Integrins are described to modulate basic synaptic function and plasticity, and to modulate the activity of the two major excitatory ionotrophic receptor subclass, NMDA and AMPA receptors. We further addressed the role of integrin activation in the normal excitatory synaptic function by utilizing in vivo single-unit recordings combined with microiontophoretic drug application in the CA1 region of the rat. Cells were excited by alternating NMDA and AMPA ejection, while integrin activation was achieved by the ejection of an RGD sequence containing pentapeptide in low and high concentration. Low integrin activation resulted in increased NMDA and decreased AMPA induced firing rate, while high RGD concentration enhanced both types of elicited responses. The control pentapeptide, pentaglycine had no effect on NMDA or AMPA evoked firing rate in either low or high concentration. These results suggest a bidirectional, dose dependent action of integrin activation on basic synaptic transmission, which may underlie the long term synaptic plasticity changes modulated by integrins.