Mammalian neurons are anoxia-sensitive and rapidly undergo excitotoxic cell death when deprived of oxygen, mediated largely by calcium entry through N-methyl-D-aspartate receptors (NMDARs). This does not occur in neurons of the anoxia-tolerant western painted turtle, where a decrease in NMDAR currents is observed with anoxia. This decrease is dependent on a modest increase in cytosolic [Ca2+] during anoxia. The aim of this study was to determine if activation of the mitochondrial permeability transition pore (mPTP) decreases NMDAR currents through release of mitochondrial Ca2+. The data indicate that mPTP opening is sufficient to cause a decrease in NMDAR currents during normoxia and the anoxia-mediated rise in cytosolic [Ca2+] and depolarization of the mitochondrial membrane potential is due to opening of the mPTP. Furthermore, since a mitochondrial uncoupler releases additional calcium during anoxia we speculate that the mitochondrial membrane potential decreases in a regulated fashion to a new set-point.

Mammalian neurons are anoxia-sensitive and rapidly undergo excitotoxic cell death when deprived of oxygen, mediated largely by calcium entry through N-methyl-D-aspartate receptors (NMDARs). This does not occur in neurons of the anoxia-tolerant western painted turtle, where a decrease in NMDAR currents is observed with anoxia. This decrease is dependent on a modest increase in cytosolic [Ca2+] during anoxia. The aim of this study was to determine if activation of the mitochondrial permeability transition pore (mPTP) decreases NMDAR currents through release of mitochondrial Ca2+. The data indicate that mPTP opening is sufficient to cause a decrease in NMDAR currents during normoxia and the anoxia-mediated rise in cytosolic [Ca2+] and depolarization of the mitochondrial membrane potential is due to opening of the mPTP. Furthermore, since a mitochondrial uncoupler releases additional calcium during anoxia we speculate that the mitochondrial membrane potential decreases in a regulated fashion to a new set-point.

No description available.

Glucose entry into the brain and its subsequent metabolism to L-lactate, regulated by astrocytes, plays a major role in synaptic plasticity and memory formation. A recent study has shown that L-lactate produced by the brain upon stimulation of glycolysis, and glycogen-derived L-lactate from astrocytes and its transport into neurons, is crucial for memory formation. A recent study revealed the molecular mechanisms that underlie the role of L-lactate in neuronal plasticity and long-term memory formation. L-lactate was shown to induce a cascade of molecular events via modulation of redox-sensitive N-Methyl-D-aspartate (NMDA) receptor activity that was mimicked by nicotinamide adenine dinucleotide hydride (NADH) co-enzyme. This indicated that changes in cellular redox state, following L-lactate transport inside the cells and its subsequent metabolism, production of NADH, and favouring a reduced state are the key effects of L-lactate. Therefore, we are investigating the role of L-lactate in modulating NMDA receptor function via redox modulatory sites. Accordingly, crucial redox-sensitive cysteine residues, Cys320 and Cys87, of the NR2A NMDA receptor subunit are mutated using site-directed mutation, transfected, and expressed in HEK293 cells. This cellular system will then be used to characterise and monitor its activity upon Llactate stimulation, compared to the wild type. This will be achieved by calcium imaging, using fluorescent microscopy. Our data shows that L-lactate potentiated NMDA receptor activity and increased intracellular calcium influx in NR1/NR2A wild type compared to the control condition (WT NR1/NR2A perfused with (1μM) glutamate and (1μM) glycine agonist only), showing faster response initiation and slower decay rate of the calcium signal to the baseline. Additionally, stimulating with L-lactate associated with greater numbers of cells having high fluorescent intensity (peak amplitude) compared to the control. Furthermore, L-lactate rescued the mutated NMDA NR1/NR2A C320A C87A receptor response that showed altered activity upon mutation up to the control level. Future experiments need to be carried out on different redox-sensitive residues of various NMDA receptor subunits to reveal the exact molecular mechanisms of L-lactate.

No description available.

The role of NMDAR subtypes in synaptic plasticity is very controversial, partially caused by the lack of specific GluN2A containing NMDA receptor (GluN2AR) antagonists. Here we took a novel approach to selectively modulate NMDAR subtype activity and investigated its role in the induction of plasticity. Whole cell recording in both acutely isolated CA1 cells and hippocampal slices demonstrated that pituitary adenylate cyclase activating peptide 1 receptors (PAC1 receptors), which are Gαq coupled receptors, selectively recruited Src kinase and enhanced currents mediated by GluN2ARs. In addition, biochemical experiments showed that the activation of PAC1 receptors phosphorylated GluN2ARs specifically. In contrast, vasoactive intestinal peptide receptors (VPAC receptors), which are Gαs coupled receptors, selectively stimulated Fyn kinase, potentiated currents mediated by GluN2B containing NMDARs (GluN2BRs). Furthermore, dopamine D1 receptor activation (another Gαs coupled receptor) specifically phosphorylated GluN2BRs. Interestingly, field recording experiments showed that PAC1 receptor activation lowered the threshold for LTP whilst LTD was enhanced by dopamine D1 receptor activation. In conclusion, the activity of GPCRs can signal through different pathways to selectively modulate absolute contribution of GluN2ARs versus GluN2BRs in CA1 neurons via Src family kinases. Furthurmore, Epac, activated by some Gαs coupled receptors, also modulated NMDAR currents via a PKC/Src dependent pathway, but whether it selectively modulates NMDAR subtypes, and has capacity to change the induction of plasticity, requires further study. By this means, we can investigate the role of NMDAR subtypes in the direction of synaptic plasticity by selectively modulating the activity of GluN2ARs or GluN2BRs. In addition, based on my work, some interfering peptides and drugs can be designed and used to selectively inhibit the activity of GluN2BRs and GluN2ARs by interrupting Fyn- and Src - mediated signaling cascade respectively. It will provide new candidate drugs for the treatment of some neurological diseases such as Alzheimer disease (AD) and schizophrenia.

N-Methyl-D-aspartate receptors (NMDARs) are a principal subtype of excitatory ligandgated ion channel with prominent roles in physiology and disease in the mammalian central nervous system (CNS). Activation of NMDARs requires binding of both glutamate and glycine. Apart from its co-agonist action, glycine can also prime NMDARs for subsequent internalization upon binding of both glutamate and glycine. However, the molecular basis responsible for mediating and regulating glycine priming and NMDAR endocytosis is largely unknown. In my thesis, I discovered the principle that although NMDAR gating and priming share a common requirement for glycine binding, the molecular constraints for gating are distinct from those for priming through two mutations of the glycine binding site in GluN1 subunit of the NMDAR that, while maintaining gating of NMDARs, eliminate glycine priming of the receptors. One of the molecular signatures of glycine priming is recruitment of the endocytic adaptor protein AP-2. I have characterized the two regions in GluN2 subunits required for enhanced AP-2 association. This unexpected result suggests binding of glycine initiates a conformational change transmitted from GluN1 to GluN2 allowing for docking of endocytic machinery. Furthermore, I have discovered that naturally occurring splice variants of GluN1 subunit, containing a 21 amino acid sequence in the N-terminus domain (N1) cassette, abrogate glycine stimulated AP-2 recruitment and glycine-primed NMDAR internalization. These findings imply that there are distinct populations of native NMDARs in the CNS – those lacking N1 that show glycine-primed internalization and those containing N1 that are not primable. Collectively, my thesis work demonstrates a dramatic all-or-none priming effect with splice variants of NMDARs, a highly unexpected discovery providing novel insight into the molecular mechanisms and physiological role of glycine priming. Ultimately, elucidating principles and mechanisms of glycine priming lay the foundation for new types therapeutic approaches for CNS disorders, approaches without the deleterious consequences of directly blocking NMDARs.

NMDA receptor signaling is required for proper synapse formation, maintenance, plasticity and function. Dysregulation of the NMDA receptor has been implicated in pathophysiology of schizophrenia, which has an adult onset of symptoms. NMDA receptor deficient mice were utilized to assess the developmental consequences of NMDA receptor hypofunction. Locomotor activity was elevated throughout development; however, deficits in social interaction and working memory only manifest in adulthood and did not progress with age. Age-dependent deficits in neuron synapse biology were also detected; postsynaptic spine number was normal in juveniles, decreased post-adolescence, and progressively declined in adulthood. To investigate possible molecular mechanisms underlying the observed changes in spine number, protein levels of RhoGTPases and their downstream effectors were examined. Significant changes in Rac1 and downstream effectors were detected at different developmental stages. These studies provide clarification of the temporal sequence of events and mechanisms by which NMDA receptor dysfunction affects neurodevelopment.

NMDA receptor signaling is required for proper synapse formation, maintenance, plasticity and function. Dysregulation of the NMDA receptor has been implicated in pathophysiology of schizophrenia, which has an adult onset of symptoms. NMDA receptor deficient mice were utilized to assess the developmental consequences of NMDA receptor hypofunction. Locomotor activity was elevated throughout development; however, deficits in social interaction and working memory only manifest in adulthood and did not progress with age. Age-dependent deficits in neuron synapse biology were also detected; postsynaptic spine number was normal in juveniles, decreased post-adolescence, and progressively declined in adulthood. To investigate possible molecular mechanisms underlying the observed changes in spine number, protein levels of RhoGTPases and their downstream effectors were examined. Significant changes in Rac1 and downstream effectors were detected at different developmental stages. These studies provide clarification of the temporal sequence of events and mechanisms by which NMDA receptor dysfunction affects neurodevelopment.

Introduction and Objectives: The PDGFβ receptor and its ligand, PDGF-BB, are expressed throughout the central nervous system (CNS), including the hippocampas. Several reports confirm that PDGFβ receptors are neuroprotective against N-methyl-D-asparate (NMDA)-induced cell death in hippocampal neurons. NMDA receptor dysfunction is important for the expression of many symptoms of mental health disorders such as schizophrenia. The serotonin (5-HT) type 7 receptor was the most recent of the 5-HT receptor family to be identified and cloned. 5-HT receptors interact with several signaling systems in the CNS including receptors activated by the excitatory neurotransmitter glutamate such as the NMDA receptor. Although there is extensive interest in targeting the 5-HT7 receptor with novel therapeutic compounds, the function and signaling properties of 5-HT7 receptors in neurons remains poorly characterized. Methods: The SH-SY5Y neuroblastoma cell line, primary hippocampal cultures, and hippocampal slices were treated with 5-HT7 receptor agonists and antagonists. Western blotting was used to measure PDGFß receptor expression and phosphorylation as well as NMDA receptor subunit expression and phosphorylation levels. Real-time RT-PCR was used to measure mRNA level of PDGFß receptor in neuronal cultures. Cell death assays (MAP2, MTT) were used to measure the neuroprotective effects of 5-HT7 and PDGFß receptor activation. Results: My research involved elucidating the molecular mechanisms of neuroprotection after 5-HT7-induced PDGFß receptor upregulation. I demonstrated that 24 h treatment with the selective 5-HT7 receptor agonist, LP 12, increased not only the expression but also the activation of PDGFß receptors as measured by the phosphorylation of tyrosine 1021, the phospholipase Cγ binding site. Activation of the 5-HT7 receptor also selectively changed the expression and phosphorylation state of the NR2B subunit of the NMDA receptor. Activation of 5-HT7 receptors was neuroprotective against NMDA-induced toxicity in primary hippocampal neurons and this effect required PDGFß receptor kinase activity. Thus, long-term (24 h) activation of 5-HT7 receptors was neuroprotective via increasing the expression of a negative regulator of NMDA activity, the PDGFß receptor. In contrast, acute activation (5-30 min) of 5-HT7 receptor increased NMDA evoked current and altered NMDA receptor subunit phosphorylation in hippocampal neurons in a manner that was different from what we observed in our 24 h experiments. Conclusions: I identified two 5-HT7 receptor to NMDA receptor pathways: acute activation of the receptor increased NMDA-evoked currents whereas long-term 5-HT7 agonist treatment prevented NMDA-induced excitotoxicity in a PDGFß receptor-dependent manner. This research is significant in the ongoing advances for the treatment of mental heath disorders, such as schizophrenia and depression, that involve the 5-HT, glutamate, and neuronal growth factor systems.