Investigating the role of HDAC4 in Drosophila neuronal function : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (PhD) in Genetics at Massey University, Manawatū, New Zealand
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Date
2022
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Massey University
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Abstract
HDAC4 plays an essential role in brain functions including neurodevelopment and memory formation, and increased levels of HDAC4 have also been associated with neurodegenerative disorders including Alzheimer’s disease.
Histone deacetylases are enzymes that are traditionally known to regulate gene expression in the nucleus, however in neurons, HDAC4 shuttles between the nucleus and cytoplasm with a predominant distribution in the cytoplasm. Although studies have identified potential differences in subcellular function in which accumulation of nuclear HDAC4 has been shown to promote neurodegeneration, while cytoplasmic HDAC4 is neuroprotective, the mechanistic pathways through which it acts are still unknown. Therefore, this project aimed to determine the importance of nuclear and cytoplasmic pools of HDAC4 to the neurological functions of Drosophila melanogaster, as well as to determine the domains within the protein that are required for its function(s). This was carried out by expressing HDAC4 with mutations that resulted in altered subcellular distribution or carrying mutations in binding domain/motifs that have previously been shown to be important for HDAC4 function.
Increased expression of wild-type HDAC4 disrupted development of the retina and the mushroom body (MB, a brain structure derived from Kenyon cells which are crucial for learning and memory), and expression of each mutant revealed the importance of specific domains/motifs to HDAC4 function in these tissues. Of interest, impairments to MB formation were exacerbated by mutation of the ankyrin-binding site and by mutation of serine residues that promote nuclear exit when phosphorylated (i.e. resulting in restriction to the nucleus). Mutation of the MEF2-binding site ameliorated these phenotypes, suggesting that HDAC4 acts through MEF2 to regulate MB development. However, while deacetylase activity was found to be dispensable in the MB, an active deacetylase domain was required in order for the phenotype to manifest in the retina, and mutation of the MEF2-binding site had no impact on the deficits caused by nuclear restriction of HDAC4 and mutation of the ankyrin-binding domain. Together these data indicate that HDAC4 acts through varying mechanism(s) depending on the cell type.
Transcriptional changes in the Drosophila brain resulting from the expression of HDAC4 or its mutant variants was also explored using RNA-Seq. However only wild-type HDAC4 resulted in a large number of differentially expressed genes and the low level of differential gene expression in HDAC4 variants suggests that non-transcriptional processes may be involved in the induction of phenotypes caused by expression of these mutants. Additionally, further analysis of genes that were differentially regulated revealed a number of processes related to mitochondrial energy production. These findings have provided new insights into the role of HDAC4 in Drosophila neurodevelopment which opens up additional research avenues to focus on in the future.
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Figures 1.12 & 6.11 were re-used with the respective publisher's permission.
Keywords
Drosophila melanogaster, Nervous system, Histone deacetylase, Nervous system, Diseases, Genetic aspects