Gábor Juhász

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.

Overexpression of Hsp27 ameliorates symptoms of Alzheimer's disease in APP/PS1 mice

Hsp27 belongs to the small heat shock protein family, which are ATP-independent chaperones. The most important function of Hsp27 is based on its ability to bind non-native proteins and inhibit the aggregation of incorrectly folded proteins maintaining them in a refolding-competent state. Additionally, it has anti-apoptotic and antioxidant activities. To study the effect of Hsp27 on memory and synaptic functions, amyloid-β (Aβ) accumulation, and neurodegeneration, we generated transgenic mice overexpressing human Hsp27 protein and crossed with APPswe/PS1dE9 mouse strain, a mouse model of Alzheimers disease (AD). Using different behavioral tests, we found that spatial learning was impaired in AD model mice and was rescued by Hsp27 overexpression. Electrophysiological recordings have revealed that excitability of neurons was significantly increased, and long-term potentiation (LTP) was impaired in AD model mice, whereas they were normalized in Hsp27 overexpressing AD model mice. Using anti-amyloid antibody, we counted significantly less amyloid plaques in the brain of APPswe/PS1dE9/Hsp27 animals compared to AD model mice. These results suggest that overexpression of Hsp27 protein might ameliorate certain symptoms of AD.

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.

Amyloid-β1-42 Disrupts Synaptic Plasticity by Altering Glutamate Recycling at the Synapse

Alzheimers disease (AD) is the most prevalent form of neurodegenerative disorders characterized by neuritic plaques containing amyloid-β peptide (Aβ) and neurofibrillary tangles. Evidence has been reported that Aβ(1-42) plays an essential pathogenic role in decreased spine density, impairment of synaptic plasticity, and neuronal loss with disruption of memory-related synapse function, all associated with AD. Experimentally, Aβ(1-42) oligomers perturb hippocampal long-term potentiation (LTP), an electrophysiological correlate of learning and memory. Aβ was also reported to perturb synaptic glutamate (Glu)-recycling by inhibiting excitatory-amino-acid-transporters. Elevated level of extracellular Glu leads to activation of perisynaptic receptors, including NR2B subunit containing NMDARs. These receptors were shown to induce impaired LTP and enhanced long-term depression and proapoptotic pathways, all central features of AD. In the present study, we investigated the role of Glu-recycling on Aβ(1-42)-induced LTP deficit in the CA1. We found that Aβ-induced LTP damage, which was mimicked by the Glu-reuptake inhibitor TBOA, could be rescued by blocking the NR2B subunit of NMDA receptors. Furthermore, decreasing the level of extracellular Glu using a Glu scavenger also restores TBOA or Aβ induces LTP damage. Overall, these results suggest that reducing ambient Glu in the brain can be protective against Aβ-induced synaptic disruption.

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.

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.

Functional changes in transcriptomes of the prefrontal cortex and hippocampus in a mouse model of anxiety.

Anxiety is a multi-etiology disorder influenced by both genetic background and environment. To study the impact of a genetic predisposition, we developed a novel mouse model of anxiety using a combination of crossbreeding and behavioral selection. Comparison of the transcriptomes from the prefrontal cortex and hippocampus of anxious and control mice revealed that the numbers of significantly up- and down-regulated genes were modest, comprising approximately 2% of the tested genes. Functional analysis of the significantly altered gene sets showed that functional groups such as nervous system development, behavior, glial cell differentiation and synaptic transmission were significantly enriched among the up-regulated genes, whereas functional groups such as potassium ion transport, Wnt signaling and neuropeptidergic signaling were significantly enriched among the down-regulated genes. Many of the identified genes and functional groups have been previously linked to the molecular biology of anxiety, while several others, such as transthyretin, vasoactive intestinal polypeptide and various potassium ion channels, are novel or not as well described in this context. Supporting the gene expression data, we also found increased excitability in the hippocampi of anxious mice, which can be a phenotypic result of decreased potassium channel density. Our transcriptome screen showed that the initiation and/or effect of anxiety involve multiple pathways and cellular processes. The identified novel genes and pathways could be involved in the molecular pathogenesis of anxiety and provide potential targets for further drug development.