Investigating the role of HDAC4 subcellular distribution in Drosophila development and memory : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Biochemistry at Massey University, Manawatū, New Zealand

Abstract

The class IIa histone deacetylase HDAC4 has been previously demonstrated to play an essential role in brain development, learning and memory. However, the molecular pathways through which it acts are unknown. HDAC4 undergoes activity-dependent nucleocytoplasmic shuttling, disruption of the balance of nuclear and cytoplasmic HDAC4 has been identified as a factor in developmental and neurodegenerative disorders. This project used Drosophila melanogaster as a model to investigate the effects of altered subcellular distribution of HDAC4 on neural development and memory formation through the overexpression of Drosophila HDAC4 and wild-type human HDAC4 (hHDAC4), as well as nuclear- and cytoplasm-localising mutants of hHDAC4 named 3SA and L175A, respectively. The nuclear or cytoplasmic abundance of HDAC4 was adjusted by expressing the mutants during development or in adult flies. It was established that increased nuclear abundance of hHDAC4 in the brain impaired long-term memory and development, whereas increasing the cytoplasmic abundance did not. Further investigation showed that, contrary to vertebrate models, HDAC4 does not appear to repress memory in Drosophila through inactivation of MEF2 or CREB. Investigation of the transcriptomic changes induced by nuclear and cytoplasmic HDAC4 via RNASeq on RNA isolated from fly heads showed that L175A unexpectedly up-regulates the expression of genes in transcription and DNA synthesis. The relatively low number of transcriptional changes induced by 3SA suggested that it may be acting through largely transcriptionally independent means to impair memory and development in Drosophila. The localisation of HDAC4 to punctate foci in nuclei, potentially forming protein aggregates similar to Marinesco bodies seen in Parkinson’s Disease warrants further investigation. This project has shown that nuclear but not cytoplasmic HDAC4 impairs development and memory in Drosophila. Furthermore, cytoplasmic HDAC4 may play a role in transcriptional regulation of neurons, possibly regulation metabolic activity, suggesting that the activity-dependent nucleocytoplasmic shuttling of HDAC4 may not be primarily to remove HDAC4 from the nucleus and but instead to return HDAC4 to the cytoplasm.