The N-methyl-D-aspartate (NMDA) receptors are highly regulated ligand-gated ion channels, which are affected by many substrates. Overactivation of the NMDA receptor can lead to hyperexcitability and a number of neurotoxic effects and neurological diseases. Agmatine has been demonstrated to act allosterically as an inhibitory modulator at the polyamine recognition sites of the NMDA receptor complex. The present study synthesized and evaluated a library of agmatine analogs for their ability to displace tritiated MK-801 from NMDARs in P2 membrane preparations from rat brains at ligand concentrations of 1 mM and 50 uM. A full dose-response curve was generated for the most active compounds, in the presence and absence of a pathological level of spermidine (100 uM). A forty-five member subset of arylidenamino-guanidino compounds was synthesized and all were demonstrated to be NMDA receptor inhibitory modulators in the above assay. Three of these compounds generated biphasic curves, indicating activity at two binding sites: the postulated high-affinity agmatine binding site, and a low-affinity site (perhaps the channel itself). (4-Chlorobenzylidenamino)-guanidine hydrochloride demonstrated an IC50 of 3.6 uM at the former site and 124.5 uM at the latter. Several computer models were generated to direct further synthesis. Based on the structure-activity relationship of the arylidenamino-guanidino compounds, a pharmacophore model of the agmatine binding site of the NMDAR was proposed.

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ionotropic receptors. When activated, NMDARs let extracellular sodium and calcium ions enter neurons. This calcium influx, depending on its duration, intensity and the presence of nearby signalling proteins can signal to synaptic plasticity. Additionally, physiological NMDAR activity promotes pro-survival cascades and gene transcription, whereas both lack of activation and overactivation of these receptors trigger pro-death signals. Several neurodegenerative pathologies such as stroke/ischemia and Alzheimer’s disease are thought to involve NMDAR overactivation, so-called “excitotoxicity”, but since NMDARs are important for normal neuronal physiology, potential therapeutical approaches needs to go beyond simple antagonism. Here, we studied the receptor subunit composition and the molecular cascades downstream of the receptor activation to try and isolate the pro-death pathways in NMDAR-mediated excitotoxicity. We found that the NR2 subunit composition did not dictate the type of NMDAR-mediated signals, as receptors comprised of NR2B subunits were able to signal to death, survival and plasticity. However, we also found that the intracellular tail of the NR2B subunit was more efficient at triggering neuronal death compared to the NR2A C-terminus, which suggests that different pro-death signalling complexes are associated to each subunit. Two pro-death signals, the p38 and c-Jun N-terminal kinase (JNK) cascades, are key mediators of neuronal excitotoxicity. In a non-neuronal cell line, NMDAR-mediated cell death could be reconstituted but was found to rely solely on JNK and not p38. This was due to the lack of pro-death signals from the NR2B-PDZ domain, a cytoplasmic interacting domain which forms a signalling cassette with the neuronal proteins PSD-95 and neuronal nitric oxide synthase. This PDZ-ligand recruits the p38 cascade in neurons, but was absent in non-neuronal cells. The pro-death p38 pathway could be inhibited in neurons by disrupting the PDZ domain interactions, which protects against excitotoxicity. This disruption was not affecting normal synaptic transmission, potentiation or survival signalling, suggesting that this could be a therapeutically viable avenue. Thus, this work has expanded the understanding of how NMDAR subunits and their cytoplasmic domains mediate signalling leading to a variety of cellular outcomes; a crucial point for the development of a strategy specifically targeting NMDAR- mediated pro-death signalling.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

The induction of synaptic plasticity at CA1 synapses requires NMDAR activation. Modulation of NMDAR function by various GPCRs can shift the thresholds for LTP and LTD induction and contribute to metaplasticity. Here we showed that the activity of GluN2A- and GluN2B-containing NMDARs is differentially regulated by Gαi/o-coupled, Gαq- and Gαs-coupled receptors. Furthermore, enhancing the relative function of GluN2A-to-GluNB NMDAR activity by GPCRs can alter the balance of LTP and LTD induction and contribute to metaplasticity. In CA1 neurons, activation of the Gαs-coupled D1/D5R selectively recruited Fyn kinase and enhanced GluN2B-mediated NMDAR currents. Biochemical experiments confirmed that D1/D5R stimulation activates Fyn kinase and enhances the tyrosine phosphorylation of GluN2B subunits. In contrast, activation of the Gαq-coupled PAC1R selectively recruited Src kinase to enhance the function of GluN2A-containing NMDARs. Enhancing the functional ratio of GluN2A-to-GluN2B subunits by PAC1R activation lowered the threshold for LTP induction whereas enhancing the functional ratio of GluN2B-to-GluN2A subunits by D1/D5R activation increased the threshold for LTP induction. Unexpectedly, activation of the Gαi/o-coupled mGluR2/3 enhanced NMDAR-mediated function via a previously unidentified mechanism. Inhibition of the cAMP-PKA pathway via mGluR2/3 activation resulted in activation of Src via decreased phosphorylation of its C-terminal Tyr527 by Csk. Stimulation of mGluR2/3 selectively potentiated the function of GluN2A-containing NMDARs but whether it shifted the modification threshold θm to the left requires further investigation.

The induction of synaptic plasticity at CA1 synapses requires NMDAR activation. Modulation of NMDAR function by various GPCRs can shift the thresholds for LTP and LTD induction and contribute to metaplasticity. Here we showed that the activity of GluN2A- and GluN2B-containing NMDARs is differentially regulated by Gαi/o-coupled, Gαq- and Gαs-coupled receptors. Furthermore, enhancing the relative function of GluN2A-to-GluNB NMDAR activity by GPCRs can alter the balance of LTP and LTD induction and contribute to metaplasticity. In CA1 neurons, activation of the Gαs-coupled D1/D5R selectively recruited Fyn kinase and enhanced GluN2B-mediated NMDAR currents. Biochemical experiments confirmed that D1/D5R stimulation activates Fyn kinase and enhances the tyrosine phosphorylation of GluN2B subunits. In contrast, activation of the Gαq-coupled PAC1R selectively recruited Src kinase to enhance the function of GluN2A-containing NMDARs. Enhancing the functional ratio of GluN2A-to-GluN2B subunits by PAC1R activation lowered the threshold for LTP induction whereas enhancing the functional ratio of GluN2B-to-GluN2A subunits by D1/D5R activation increased the threshold for LTP induction. Unexpectedly, activation of the Gαi/o-coupled mGluR2/3 enhanced NMDAR-mediated function via a previously unidentified mechanism. Inhibition of the cAMP-PKA pathway via mGluR2/3 activation resulted in activation of Src via decreased phosphorylation of its C-terminal Tyr527 by Csk. Stimulation of mGluR2/3 selectively potentiated the function of GluN2A-containing NMDARs but whether it shifted the modification threshold θm to the left requires further investigation.

Orientadores: Elenice Aparecida de Moraes Ferrari, Luiz Roberto Giorgetti Britto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T11:20:23Z (GMT). No. of bitstreams: 1 Sperandeo_MariaLuizaAntunes_M.pdf: 1329953 bytes, checksum: 9e4252fe270a020cdc4182cde457725e (MD5) Previous issue date: 2005 / Resumo: O presente estudo investigou os efeitos do antagonista do receptor NMDA, MK-801 na expressão do produto do zenk no hipocampo (Hp) de pombos, submetidos ao condicionamento clássico aversivo. Antes do treino, administrou-se MK-801, i.p, para o grupo condicionado MK (GCMK, n=6), salina para o grupo condicionado salina (GCS, n=6) e nenhum tratamento para os grupos-controle: randômico (GCR, n=6), contexto (GCC=7) e manipulação (GM=4). GCMK e GCS receberam três associações de som (1000-Hz, 83 dB,1 s) e choque (10 mA, 35ms) numa sessão de 20 min. Para GCR os estímulos foram aleatórios e o GCC não recebeu estímulos. O teste de re-exposição ao contexto ocorreu 24 h após o treino. A análise de freezing no treino mostrou maior ocorrência para o GCS em comparação ao GCC (p<0,05), com aumento gradual na sessão (p<0,01). No teste, GCS expressou maior ocorrência de freezing em comparação a todos os grupos (p<0,001). A expressão de zenk foi avaliada por imuno-histoquímica. O GCS teve maior número de núcleos ZENK-positivos no Hp ventral, especificamente no Hp ventro-medial, comparativamente aos outros grupos (p<0,01). A baixa ocorrência de freezing ao contexto no GCMK evidencia o efeito amnésico do MK-801. A análise da marcação de núcleos ZENK-positivos no Hp sugeriu sua ativação regionalizada na evocação de memória contextual aversiva em pombos. O presente estudo indica o envolvimento de receptores de glutamato do tipo NMDA em mecanismos sinápticos de plasticidade neural durante a evocação de memória aversiva ao contexto. Palavras-chave: condicionamento clássico aversivo, hipocampo, MK-801, antagonista dos receptores NMDA, recuperação da memória aversiva, zenk / Abstract: The present study investigated the effects of the antagonist of the glutamate NMDA receptor, MK- 801, in the activation of zenk in the hippocampus of pigeons (Hp) submitted to the classical aversive conditioning. Two groups of pigeons received MK-801 (MKG, n=6) or saline (SG, n=6) 30 min before training with tone-shock associations. The control groups received unpaired stimulation (RCG, n=6), exposure to the context (CCG=7) or manipulation alone (MG=4). During the 20 min training session MKG and SG received three sound (1000-Hz, 83 dB, 1 s) and shock associations (10 mA, 35ms). The test to the context occurred 24 hours after the training. During the training session SG animals showed more freezing as compared with CCG (p<0,05). During the test, SG expressed higher freezing than all the other groups (p<0,001). ZENK analysis was conducted with imunohistochemistry. The density of ZENK-positive nuclei in the ventral hippocampus, specifically in the ventromedial hippocampus, was higher for SG as compared to the other groups (p<0,01). The fact that the animals from the MKG expressed lower freezing to the context may be considered as indicative of an amnesic effect of the MK-801. The density of ZENK-positive nuclei in the hippocampus suggests a regional activation that may be related to the retrieval of contextual aversive memory. The present study indicates that synaptic mechanisms mediated by NMDA glutamate receptors participate in the neural plasticity related to the retrieval of contextual aversive memory / Mestrado / Fisiologia / Mestre em Biologia Funcional e Molecular

Little is known about higher order chromatin structures in the human brain and their function in transcription regulation. We employed chromosome conformation capture (3C) to analyze chromatin architecture within 700 Kb surrounding the transcription start site (TSS) of the NMDA receptor and schizophrenia susceptibility gene, GRIN2B, in human and mouse cerebral cortex. Remarkably, both species showed a higher interaction between the TSS and an intronic sequence, enriched for (KRAB) Krueppel associated Box domain binding sites and selectively targeted by the (H3K9) histone 3 lysine 9 specific methyltransferase ESET/SETDB1. Transgenic mice brain cortical nuclei over-expressing Setdb1 showed increased heterochromatin-protein 1 signal at the interacting regions coupled with decreased Grin2b expression. 3C further revealed three long distant chromatin loop interactions enriched with functional enhancer specific (H3K27Ac) histone 3 lysine 27 acetylation signal in GRIN2B expressing tissue (human cortical nuclei and Human Embryonic Kidney - HEK cells). Doxycycline-induced SETDB1 over-expression decreased 2 out of 3 loop interaction frequencies suggesting a possible SETDB1-mediated transcription repression. We also report a specific looping interaction between a region 50Kb upstream of the (GAD1) Glutamic Acid Decarboxylase – 1 gene TSS and the GAD1 TSS in human brain nuclei. GAD1 catalyzes the rate limiting step in (GABA) gamma amino-butyric acid synthesis and is quintessential for inhibitory signaling in the human brain. Clinical studies in schizophrenia brain samples reveal a decreased looping interaction frequency in correspondence with a decrease in gene expression. Our findings provide evidence for the existence of transcription relevant higher order chromatin structures in human brain.