Thesis

Functional screening for gene trap mutants involved in perineuronal net formation.

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Awarding institution
  • University of Strathclyde
Date of award
  • 2023
Thesis identifier
  • T16713
Person Identifier (Local)
  • 201879613
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Qualification Name
Department, School or Faculty
Abstract
  • A tissue’s cells function within the context of an extracellular matrix (ECM) of glycosylated and crosslinked proteins that provide structural support and adhesion, while also modulating intercellularcommunication. In the central nervous system (CNS), the ECM is vital for the complex neuronalmigration and neurite projection stages during embryonic development that create the preciseanatomies and morphologies of functioning brain circuitry.The historical discovery of a plant-derived molecule, Wisteria floribunda agglutinin (WFA),revolutionised the study of brain ECM because it binds to (and, therefore, is used to stain) onespecific form of ECM called the perineuronal net (PNN). As its name suggests, this creates a ‘cage’specifically around the soma of parvalbumin-containing interneurons. The critical role of this class ofneurons in brain activity regulation, and known involvement in specific neuropsychiatric pathologies,ignited much investigation of the PNN. It is now known that its dysfunction is associated with multipleconditions. In our laboratory, a previous cellular ‘gene trap’ genetic screen identified that the mutationof protein components of the PNN contributed to the response to lithium, a mood stabiliser treatmentfor the psychiatric disorder bipolar disorder.In this thesis, the further application of gene trap screening to search for genes encoding proteinsthat contribute directly to, or regulate, the formation of the perineuronal net on SH-SY5Yneuroblastoma cells, a commonly used model of neurons is described. The hypothesis was thatidentified genes would not only provide greater insight into the PNN structure but might offer newtargets for the treatment of CNS disorders. A ‘library’ of randomly mutated cells was created, andWFA was used to identify mutant colonies with reduced staining, indicative of PNN dysfunction.Several PNN-defective cell colonies were isolated, and their mutated genes identified using amodified polymerase chain reaction (PCR) protocol. Three genes, DCC, FAF1, and GALNTL6 wereamong those that were identified and considered the best candidates to take forward for furtheranalysis. DCC protein is the netrin-1 receptor, which has important roles in CNS development. FAF1,the FAS associated factor 1 protein, which participates in apoptosis and autophagy processes.GALNTL6 is a glycosyl transferase enzyme that modifies proteins through O-linked glycosylation –a very strong candidate in light of the substantial glycosylation that occurs to PNN proteins, andwhich is thought to be the target of WFA staining. These three proteins required validation through the generation of independent mutations/inhibitionin cells. CRISPR, siRNA and pharmacological means was used to attempt this. Multiple geneticablations failed to produce successful defective gene alleles meaning that full validation of thesethree candidate genes was not possible, and their role in PNN function remains unclarified.However, the still-unknown targets of WFA in a ‘pull-down’ assay of proteins lysed from SH-SY5Ycells was pursued-associated protein material was assessed by mass spectrometry. Among the tophits was the protein vimentin which is known to exist within the cytoplasm of the cells but also to besecreted into the ECM, where it reportedly shows post-translational modification by O-linkedglycosylation.In summary, despite failure to fully validate screen findings, these studies identified a number ofcandidates for further investigation in the context of PNN function and role in associated disease.The protein vimentin should also be pursued in terms of its potential contribution to PNN functionand as the target of the WFA stain.
Advisor / supervisor
  • Pickard, Benjamin
Resource Type
DOI

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