Taha, Fatma M H A; (2024) A multidisciplinary approach towards characterizing the mechanism of tolerance development to diazepam. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Diazepam is a positive allosteric modulator of GABAA-Rs eliciting effects including anxiolysis, sedation and muscle relaxation. Recent evidence shows a series of coordinated molecular and cellular events that take place in response to chronic diazepam treatments, which begin with dissociation of phospholipase-C-delta-1 (PLCδ1) from GABAA-Rs, ending with a reduction in levels of cell-surface receptors and inhibitory synapses. In this research, I wanted to define where PLCδ1 associates with GABAA-Rs, employing bioinformatics and computational homology modelling. Using Modeller 9v21, models of GABAA-Rs and PLCδ1 were produced and docking of these two proteins was simulated using HADDOCK and ClusPro. Both algorithms proposed a consensus of five residues located at the β3-subunit intracellular loop. Then, using site-directed mutagenesis and binding assays I aimed to scale down these five residues further, to one or two key residues which is/are most likely the site of PLCδ1 interactivity. Two residues were consistently found as likely to meet this criterion, Q778 and R780, but further studies are needed for confirmation. Furthermore, animal models of chronic diazepam treatments were produced in this research, which in addition to behavioral endpoints indicative of tolerance, were aimed at correlating protein expression consequences at the level of GABAA-R subunits, PLCδ1, and other proteins critical for diazepam tolerance. Spontaneous-locomotor-activity test and open-field test were used to track the development of behavioral tolerance in mice treated chronically with diazepam and to capture it once reached. With one paradigm, mice showed clear evidence of diazepam tolerance in behavioral endpoints but not at the level of protein expression. With the other paradigm, behavioral tolerance was not clearly attained, but an in-depth analysis is presented to explain the observed anxiety-like state of the mice treated acutely with diazepam. All in all, findings in this research drive the work further forward towards the better understanding of how tolerance to diazepam potentially develops.

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