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Title: Biological insights into inhibitory glycinergic transmission in zebrafish and mice
Author: Leacock, S. E. M.
ISNI:       0000 0004 7223 7971
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2017
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This study describes an investigation into the biological roles of selected glycine receptor (GlyR) subtypes in startle disease/hyperekplexia using zebrafish and mouse models. Firstly, I investigated the molecular basis of a novel zebrafish mutant dhx37nig1 with defective glycinergic transmission and designed morpholinos to knockdown DEAH box RNA helicase-37 and GlyR α and β subunits to recapitulate the phenotype. Secondly, using site-directed mutagenesis and molecular genetics techniques, I introduced a dominant- negative mutation (p.R271Q) common in human startle disease into vector- containing zebrafish GlyR α2 and GlyR α4 subunit cDNAs as an alternative method for targeted gene knockdown in zebrafish. Using CRISPR/Cas9 technology, I also generated a zebrafish glra2 knockout and developed a diagnostic PCR to genotype mutants with a large 26 base pair deletion. The glra2 knockout line was used to explore the role of the GlyR α2 subtype in locomotion and brain and spinal motor axon morphology. Thirdly, using bioinformatic tools, I investigated the GlyR α4 subunit in vertebrate species, looking for molecular evidence to explain why this gene is a pseudogene in humans. This revealed that, in addition to an in-frame stop codon, the human GlyR α4 subunit gene contains at least two other damaging mutations (p.E59K and p.Y204C) that were also present in ancient humans such as Denisovans. Lastly, using molecular genetic techniques, I identified candidate mutations in the shaky mouse, a mutant with an exaggerated startle response and tremors. This revealed a missense mutation causing the amino acid change p.Q177K in extracellular loop F of the GlyR α1 subunit. This shaky mutant showed defective integration into synaptic GlyRs as well as decreased current amplitudes with significantly faster decay times. My study highlights how the use of modern genetics techniques and model organisms can provide new insights into the biology of glycinergic transmission that underlies neurological disease.
Supervisor: Harvey, R. J. ; Suster, M. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available