Browsing by Author "Wilson, Sarah Jean"

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Characterising CG5846 (Peep) in Drosophila melanogaster neural function : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand
(Massey University, 2024) Wilson, Sarah Jean
Histone deacetylase 4 (HDAC4) is a transcriptional regulator that has been implicated in a number of neurodevelopmental and neurodegenerative diseases that are associated with intellectual disability, cognitive defects, and/or memory loss. Both the accumulation of nuclear HDAC4 and its loss-of-function have been linked to these conditions, therefore exploring HDAC4’s role in neuronal function is essential to understand the molecular mechanisms underlying these diseases. In Drosophila, overexpression of HDAC4 results in defects in morphogenesis of axons in the mushroom body, a structure essential for memory formation, as well as long-term memory defects and disruption to the development of the compound eye. The molecular mechanisms underlying these HDAC4-induced phenotypes are currently unknown. RNA-sequencing on fly heads in which HDAC4 was overexpressed has previously been performed and showed few genes were transcriptionally regulated by HDAC4. In addition, an enhancer/suppressor rough eye phenotype screen has also been performed which identified a number of genes that interact genetically in the same molecular pathway as HDAC4. To further investigate the molecular mechanisms underlying HDAC4 dysfunction, an RNA interference (RNAi) based candidate screen for potential HDAC4-interactors was performed, which involved quantification of developmental defects in the mushroom body and eye following RNAi knockdown of each candidate. It was hypothesised that if a phenotype resulting from RNAi knockdown was similar to that induced by HDAC4 overexpression, that candidate may function in similar molecular pathways. A single candidate-interactor was selected (CG5846, named Peep) for further investigation. On overexpression, Peep and HDAC4 co- distribute in nuclei of mushroom body neurons, however no physical interaction was detected. Furthermore, overexpression of Peep did not rescue the HDAC4-induced mushroom body or eye defects. Due to the uncharacterised nature of Peep, a thorough investigation was performed to assess the importance of Peep in survival, longevity, motor function, brain development, courtship learning and memory, and wing development. Peep was observed to be essential for survival of glial cells and for normal mushroom body development, which warrants further investigation. Reduced expression of Peep also resulted in a unique severe necrotic eye phenotype, and through this, Peep was shown to play a potential role in processes involved in regulating mitochondrial and proteasomal function, apoptosis and oxidative stress. These data provide the first documented characterisation of the functional role of Peep in Drosophila development and provide the basis for further investigation into the underlying molecular mechanisms involved in mushroom body and eye development.
Teasing apart the interaction between HDAC4 and Ankyrin2 in Drosophila neuronal function : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry, School of Fundamental Sciences, Massey University, Manawatu, New Zealand
(Massey University, 2021) Wilson, Sarah Jean
Histone deacetylase 4 (HDAC4) is a class IIa histone deacetylase that has previously been implicated in a range of neurodevelopmental and neurodegenerative diseases which involve deficits in memory and cognition. Overexpression of HDAC4 in the Drosophila brain impairs memory, therefore making Drosophila an ideal genetic model system to further investigate the molecular pathways through which HDAC4 acts. A recent genetic screen in Drosophila for genes that interact in the same molecular pathway as HDAC4 identified the cytoskeletal regulator Ankyrin2 (Ank2). The Ank2 protein plays a pivotal role in maintaining the stability and plasticity of the spectrin-actin cytoskeleton by organising the distribution of ion channels and cell adhesion molecules, which is essential to normal learning and memory formation. Both overexpression of HDAC4 and knockdown of Ank2 result in similar deficits in Drosophila brain development and long-term memory formation, suggesting that these two proteins may interact together in such processes. HDAC4 contains an N-terminal ankyrin repeat binding motif and it was hypothesised that HDAC4 interacts physically with the ankyrin repeat region at the N-terminus of Ank2, however, no physical interaction was detected via co-immunoprecipitation. Further investigation was then carried out to elucidate the nature of the genetic interaction proposed between HDAC4 and Ank2. In doing so, it was observed that nuclear accumulation of HDAC4 is required for this interaction, however, the presence of the HDAC4 ankyrin repeat binding motif is not required. This is consistent with the finding that HDAC4 does not bind Ank2 and indicates that the interaction between HDAC4 and Ank2 is indirect. It was also identified that Ank2 and HDAC4 are both required for Drosophila eye development as knockdown of Ank2 paired with overexpression of HDAC4 resulted in a severe novel "blueberry" phenotype that has not yet been characterised for these genes. Furthermore, it was observed that Ank2 was required for normal growth and morphogenesis of dendrites in the visual system, whereby both knockdown of Ank2 and overexpression of HDAC4 disrupt dendrite morphogenesis. These data provide further understanding of the roles of HDAC4 and Ank2 in Drosophila neuronal function, and the establishment of the molecular pathway in which HDAC4 and Ank2 act will be essential in unravelling additional mechanisms involved in the processes of learning and memory.