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

Abstract

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.