Fedele, Laura; (2018) Dysfunctional NMDA receptors in neurological disorders. Doctoral thesis (Ph.D), UCL (University College London).

Abstract

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that together with a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) and kainate receptors mediate the vast majority of the fast excitatory neurotransmission in the central nervous system. Given this role, any dysfunction in neurotransmission is likely to have a severe impact on brain physiology. Recent mutations have been reported in NMDAR subunits that cause patients to suffer with a variety of neurodevelopmental disorders. Here, we use multidisciplinary structural modelling, site-directed mutagenesis, electrophysiology and kinetic modelling techniques to investigate how de novo missense mutations in distinct regions of the GluN2B subunit, affect NMDAR function. We predicted that these mutations would have pathophysiological implications and we sought to examine their effects on the cellular and molecular function of NMDARs. We developed a virtually complete 3D model of the human GluN1-GluN2B receptor based on the recently solved crystal structures of the frog and rat NMDARs. The human NMDAR structure locates the positions of the residues of interest, allows deductions about their potential impact on the protein as well as provides insight into the binding sites for Mg2+ and memantine using molecular docking. The functional effects of the missense mutations were first analysed in recombinant NMDARs and revealed gain-of-function and loss-of-function phenotypes, with some lacking an overt phenotype. We selected four most profound phenotypes for study in hippocampal cultured neurons revealing how these mutations can compromise excitatory neurotransmission. In addition, we also explored the therapeutic potential of the FDA-approved channel blocker memantine both in heterologous system as well as on excitatory neurotransmission as a potential therapeutic. Overall, the results suggest strong correlations between the effects of the missense mutations with patient phenotypes. Moreover, the study indicates which pharmacotherapeutic interventions are most likely to be successful as targeted therapies.

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