Characterisation of the Redox Sensitive NMDA Receptor

Handle URI:
http://hdl.handle.net/10754/609187
Title:
Characterisation of the Redox Sensitive NMDA Receptor
Authors:
Alzahrani, Ohood ( 0000-0001-9214-9635 )
Abstract:
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.
Advisors:
Magistretti, Pierre J. ( 0000-0002-6678-320X )
Committee Member:
Fiumelli, Hubert ( 0000-0001-5136-3045 ) ; Arold, Stefan T. ( 0000-0001-5278-0668 )
KAUST Department:
Biological and Environmental Sciences and Engineering (BESE) Division; Bioscience
Program:
Bioscience
Issue Date:
May-2016
Type:
Thesis
Appears in Collections:
Theses; Biological and Environmental Sciences and Engineering (BESE) Division

Full metadata record

DC FieldValue Language
dc.contributor.advisorMagistretti, Pierre J.en
dc.contributor.authorAlzahrani, Ohooden
dc.date.accessioned2016-05-12T13:54:03Zen
dc.date.available2016-05-12T13:54:03Zen
dc.date.issued2016-05en
dc.identifier.urihttp://hdl.handle.net/10754/609187en
dc.description.abstractGlucose 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.en
dc.language.isoenen
dc.subjectCharacterizationen
dc.subjectRedox-sensitiveen
dc.subjectNMDA receptoren
dc.titleCharacterisation of the Redox Sensitive NMDA Receptoren
dc.typeThesisen
dc.contributor.departmentBiological and Environmental Sciences and Engineering (BESE) Divisionen
dc.contributor.departmentBioscienceen
thesis.degree.grantorKing Abdullah University of Science and Technologyen_GB
dc.contributor.committeememberFiumelli, Huberten
dc.contributor.committeememberArold, Stefan T.en
thesis.degree.disciplineBioscienceen
thesis.degree.nameMaster of Scienceen
dc.person.id132594en
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