Thesis
Functional screening for gene trap mutants involved in perineuronal net formation.
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2023
- Thesis identifier
- T16713
- Person Identifier (Local)
- 201879613
- Qualification Level
- 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 intercellular communication. In the central nervous system (CNS), the ECM is vital for the complex neuronal migration and neurite projection stages during embryonic development that create the precise anatomies 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) one specific 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 of neurons 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 multiple conditions. In our laboratory, a previous cellular ‘gene trap’ genetic screen identified that the mutation of protein components of the PNN contributed to the response to lithium, a mood stabiliser treatment for the psychiatric disorder bipolar disorder. In this thesis, the further application of gene trap screening to search for genes encoding proteins that contribute directly to, or regulate, the formation of the perineuronal net on SH-SY5Y neuroblastoma cells, a commonly used model of neurons is described. The hypothesis was that identified genes would not only provide greater insight into the PNN structure but might offer new targets for the treatment of CNS disorders. A ‘library’ of randomly mutated cells was created, and WFA 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 a modified polymerase chain reaction (PCR) protocol. Three genes, DCC, FAF1, and GALNTL6 were among those that were identified and considered the best candidates to take forward for further analysis. 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, and which is thought to be the target of WFA staining. These three proteins required validation through the generation of independent mutations/inhibition in cells. CRISPR, siRNA and pharmacological means was used to attempt this. Multiple genetic ablations failed to produce successful defective gene alleles meaning that full validation of these three 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-SY5Y cells was pursued-associated protein material was assessed by mass spectrometry. Among the top hits was the protein vimentin which is known to exist within the cytoplasm of the cells but also to be secreted into the ECM, where it reportedly shows post-translational modification by O-linked glycosylation. In summary, despite failure to fully validate screen findings, these studies identified a number of candidates 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 function and as the target of the WFA stain.
- Advisor / supervisor
- Pickard, Benjamin
- Resource Type
- DOI
Relations
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PDF of thesis T16713 | 2023-10-03 | Public | Download |