Alcohol affects many neurochemical processes, causing long-lasting changes in both the adult and developing brain. The Jak-Stat transcriptional activation pathway plays a role in the control of neuronal proliferation, survival and differentiation, but the effects of ethanol on the system have not been fully elucidated. The goal of this project was to define the effects of acute and subchronic ethanol exposure on the expression of proteins in the Jak-Stat pathway, using cultured NG108-15 cells, and in addition, to test the hypothesis that these effects are mediated through the NMDA receptor. I found that ethanol dose-dependently decreased Jak2 and Stat3 following subchronic exposure of NG108-15 in culture. Acute ethanol exposure caused a dose-dependent decrease in Stat3 protein levels. Incubation with MK-801 or ketamine, two noncompetitive NMDA receptor antagonists, or the receptor agonist NMDA, produced dose-dependent decreases in Stat3 protein as well.

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.

No description available.

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.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

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

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: During glutamate excitotoxicity, overstimulation of glutamate receptors leads to sustained elevation in cytosolic Ca2+ ([Ca2+]c), or delayed Ca2+ dysregulation (DCD), which is causally linked to cell death. There are two major hypothetical mechanisms for DCD: the continuous activation of N-methyl-D-aspartate-subtype of the ionotropic glutamate receptors (NMDAR) and the reversal of the plasmalemmal Na+/Ca2+ exchanger. However, the contribution of each of these mechanisms in DCD is not completely established. Major results: Neurons exposed to excitotoxic glutamate produced DCD, an increase in cytosolic Na+ ([Na+]c), and plasma membrane depolarization. MK801 and memantine, noncompetitive NMDAR inhibitors, added after glutamate, completely prevented DCD; however AP-5, a competitive NMDAR inhibitor, failed to do so. The NMDAR inhibitors had no effect on lowering elevated [Na+]c or on restoring plasma membrane potential, which are conditions suggesting NCXrev could be involved. In experiments inducing NCXrev, MK801 and memantine completely inhibited Ca2+ dysregulation after glutamate while AP-5 did not. Inhibition of NCXrev, either with KB-R7943 or by preventing the increase in [Na+]c, failed to avert DCD. However, NCXrev inhibition combined with NMDAR blocked by AP-5 completely prevented DCD. Overall, these data suggested that both NMDAR and NCXrev are essential for glutamate-induced DCD, and inhibition of only one mechanism is insufficient to prevent collapse of calcium homeostasis. Based on the data above, we investigated a NMDA receptor antagonist currently in clinical trials for reducing the effects of glutamate excitotoxicity, ifenprodil. Ifenprodil is an activity-dependent, NMDAR inhibitor selective for the NR2B subunit. We found that ifenprodil not only inhibited the NR2B-specific NMDAR, but also inhibited NCXrev. If ifenprodil is combined with PEAQX, a NMDAR inhibitor selective for the NR2A subunit, low concentrations of both inhibitors completely prevent DCD. Conclusion: The inhibition of a single Ca2+ influx mechanism is insufficient in preventing DCD, which requires simultaneous inhibition of both the NMDAR and NCXrev. These findings are critical for the correct interpretation of the experimental results obtained with these inhibitors and for better understanding of their neuroprotective actions.

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.