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Title: A network informed investigation of evolutionary conserved effectors of synaptic function
Author: Dawson, Bronwyn
ISNI:       0000 0004 6422 8514
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Neuronal function is a highly complex process, and model organisms are required to elucidate novel pathway components that may play a role in neurological disease. High-throughput targeted RNAi screens were performed on worm strains sensitive to neuronal RNAi, in order to assess the functional consequences of changing gene expression on synaptic function, as assessed by the relative changes in sensitivity to the acetylcholinesterase inhibitor aldicarb. A one-step binary aldicarb-sensitivity protein interaction network was constructed, using all genes with a known aldicarb sensitivity phenotype as core nodes. Connectivity and betweenness to genes known to cause a change in aldicarb sensitivity was measured. 19 genes highly connected and 17 high betweenness genes displayed a change in aldicarb sensitivity, with 8 having both high betweenness and connectivity. Previous microarray studies on the effects of sodium valproate were carried out on wild type worms (Munasinghe, 2015), and suggested that valproate could conditionally influence synaptic function at neuromuscular junctions. To investigate this relationship further genes that exhibited dose dependent changes in gene expression were identified and clustered within the one-step aldicarb betweenness network using a module prediction algorithm. Proteins within functional modules that were not previously known to exhibit aldicarb sensitivity were systematically screened, and 16 genes were shown to cause a significant change in the rate of aldicarb-induced paralysis, including sel-12 and dhhc-2, a palmitoyl-acyl transferase. Data from valproate dose response microarrays were re-analyzed and DAVID, Ingenuity and WebGestalt analyses were performed on the subset of genes showing valproate-induced changes in gene expression. Several known disease associations were identified, including Alzheimer's disease, epilepsy and Huntington's disease. Microarray data from other available valproate sensitivity studies were also analyzed, and the overlap in genes with the worm micro array dataset was assessed. This analysis suggests that changes in neuronal development and differentiation, Wnt signaling and cholinergic transmission may all be functionally affected by exposure to valproate. Dhhc-2 is a member of the palmitoyl-acyl transferase family, and the rest of the family was systematically tested using aldicarb-sensitivity assays. The dhhc-13 mutant was shown to cause hypersensitivity and the dhhc-14 showed resistance to aldicarb, suggesting mechanisms of reciprocal conditional regulation. Hypersensitivity to aldicarb was also observed when dhhc-1 and dhhc-5 were knocked down, while dhhc-3, -11, and spe-10 genetic knockdown led to resistance. The dhhc-2 mutant and RNAi knockdown both caused enhanced paralysis when treated with 15mM valproate, suggesting a functional effect of the drug at neuromuscular junctions. A selection of human PATs were analyzed in the Y2H library screens using cytoplasmic domains of DHHC5, 5, 13 and 17. DHHC5CT, 17N and 13N revealed 11, 13 and 11 interaction partners, respectively. Membrane based Y2H library screening was also carried out, and 17 novel interaction partners of full length DHHC9 were identified, including reticulin and selenoprotein K. These screens revealed interesting novel interaction partners, which could provide new insights into the mechanisms by which PAT family members may differentially affect neuromuscular function.
Supervisor: Sanderson, C. ; Barclay, J. ; Stagi, M. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral