Protein palmitoylation (also known as S-acylation) is a widespread post-translational modification that regulates the trafficking and function of a diverse array of proteins. This modification is catalysed by a family of twenty-three zDHHC enzymes that exhibit both specific and overlapping substrate interactions. Mutations in the gene encoding zDHHC9 cause mild-to-moderate intellectual disability, seizures, speech and language impairment, hypoplasia of the corpus callosum and reduced volume of sub-cortical structures. In this thesis, behavioural phenotyping, magnetic resonance imaging (MRI), isolation of S- acylated proteins and metabolomics were conducted to investigate the effect of knock- out (KO) of the Zdhhc9 gene in mice in a C57BL/6 genetic background.As a first step, end-point and quantitative Polymerase Chain reaction (PCR) were used to confirm KO of Zdhhc9 expression. Interestingly, although the mutant mice had ablated expression of mRNA encoding full-length zDHHC9, the mice did express reduced levels of a shorter Zdhhc9 transcript. However, immunoblotting analysis suggested that this shorter mRNA fragment did not lead to expression of a stable truncated form of zDHHC9 and further confirmed loss of zDHHC9 expression. Thus, the mouse line appears to be a true knock-out.The Zdhhc9 KO male mice exhibit a range of abnormalities compared with their wild-type littermates: altered behaviour in the open-field test, elevated plus maze and acoustic startle test that is consistent with a reduced anxiety level; a reduced hang time in the hanging wire test that suggests underlying hypotonia but which may also be linked to reduced anxiety; deficits in the Morris water maze test of hippocampal-dependent spatial learning and memory; and a 36% reduction in corpus callosum volume revealed by MRI. Surprisingly, palmitoylation of several important pre- and post-synaptic proteins was not disrupted in either whole brain or hippocampus of Zdhhc9 KO mice, including H-Ras which has previously been proposed to be a key substrate of zDHHC9. This suggests that other unidentified substrates of zDHHC9 are linked to the observed changes.Metabolomic profiling identified the pathways of tryptophan metabolism and of phenylalanine, tyrosine and tryptophan biosynthesis as significantly dysregulated in Zdhhc9 KO mice. Overall, this study highlights a key role for zDHHC9 in brain development and behaviour and supports the utility of the Zdhhc9 KO mouse line to investigate molecular and cellular changes linked to intellectual disability and other deficits in the human population.
|Date of Award||8 Oct 2018|
- University Of Strathclyde
|Sponsors||University of Strathclyde|
|Supervisor||Luke Chamberlain (Supervisor) & Judith Pratt (Supervisor)|