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Subject: search.filters.subject.Histone deacetylase

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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.
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    Ankyrin2 is essential for neuronal morphogenesis and long-term courtship memory in Drosophila.
    (BioMed Central Ltd, 2023-05-16) Schwartz S; Wilson SJ; Hale TK; Fitzsimons HL
    Dysregulation of HDAC4 expression and/or nucleocytoplasmic shuttling results in impaired neuronal morphogenesis and long-term memory in Drosophila melanogaster. A recent genetic screen for genes that interact in the same molecular pathway as HDAC4 identified the cytoskeletal adapter Ankyrin2 (Ank2). Here we sought to investigate the role of Ank2 in neuronal morphogenesis, learning and memory. We found that Ank2 is expressed widely throughout the Drosophila brain where it localizes predominantly to axon tracts. Pan-neuronal knockdown of Ank2 in the mushroom body, a region critical for memory formation, resulted in defects in axon morphogenesis. Similarly, reduction of Ank2 in lobular plate tangential neurons of the optic lobe disrupted dendritic branching and arborization. Conditional knockdown of Ank2 in the mushroom body of adult Drosophila significantly impaired long-term memory (LTM) of courtship suppression, and its expression was essential in the γ neurons of the mushroom body for normal LTM. In summary, we provide the first characterization of the expression pattern of Ank2 in the adult Drosophila brain and demonstrate that Ank2 is critical for morphogenesis of the mushroom body and for the molecular processes required in the adult brain for the formation of long-term memories.
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    Investigating HDAC4 aggregation in a Drosophila model of neuronal development : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2024-06-21) Hawley, Hannah Rose
    Histone deacetylase four (HDAC4) is essential in neuronal development and function, and dysregulation of HDAC4 has been observed in a number of neurodevelopmental and neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases. In particular, its aberrant nuclear accumulation is a common feature among these diseases, and it has been observed that upon upregulation or accumulation in the nucleus, HDAC4 forms punctate foci in neuronal nuclei. Previous research in a Drosophila model determined that overexpression of HDAC4 disrupted both neuronal development and long-term memory, and this was largely mediated by the nuclear pool of HDAC4. Based on these data, it was hypothesised that aggregates of HDAC4 are responsible for the neurotoxicity that leads to disrupted neurodevelopment and memory. Therefore, this study aimed to determine whether the presence of HDAC4 nuclear aggregates correlated with neurodevelopmental deficits in a Drosophila model of neurodevelopment, and if so, how they mediate their toxic effects. The N-terminus of HDAC4 forms homo-oligomers in solution, and it was hypothesised that full-length HDAC4 similarly oligomerises, and that this is required for its aggregation in neuronal nuclei. Mutations predicted to prevent oligomerisation were introduced into the N- terminus of HDAC4 and were shown to significantly reduce aggregation of HDAC4 in Drosophila neurons. Furthermore, their presence also reduced the severity of HDAC4 overexpression-induced impairments in neurodevelopment. Conversely, stabilisation of oligomerisation increased aggregation and the severity of neurodevelopmental phenotypes, together indicating that aggregation positively correlates with the severity of neurodevelopmental deficits. HDAC4 aggregates have been previously shown to sequester the transcription factor MEF2, and further investigation revealed that the presence of MEF2 stabilised aggregation and increased the severity of defects in neuronal development. Importantly, targeting the interaction between HDAC4 and MEF2 reduced the severity of these defects. Other than MEF2, the composition of HDAC4 aggregates is unknown, and therefore immunoprecipitation-coupled mass spectrometry was performed on nuclear HDAC4 to identify candidate interactors of aggregates. This revealed a number of proteins with roles in neuronal development and function, as well as those involved in splicing and protein homeostasis, suggesting that aggregates may be impairing these processes to mediate toxicity. Together these data indicate that nuclear aggregation of HDAC4 impairs neurodevelopment, and may constitute a novel biomarker of disease or therapeutic target. Given the overlap in aetiology between neurodevelopmental and neurodegenerative diseases, further investigation of whether HDAC4 aggregation contributes to the severity and/or progression of neurodegenerative disorders is warranted.
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    Deciphering the roles of subcellular distribution and interactions involving the MEF2 binding region, the ankyrin repeat binding motif and the catalytic site of HDAC4 in Drosophila neuronal morphogenesis
    (BioMed Central Ltd, 2024-12) Tan WJ; Hawley HR; Wilson SJ; Fitzsimons HL
    BACKGROUND: Dysregulation of nucleocytoplasmic shuttling of histone deacetylase 4 (HDAC4) is associated with several neurodevelopmental and neurodegenerative disorders. Consequently, understanding the roles of nuclear and cytoplasmic HDAC4 along with the mechanisms that regulate nuclear entry and exit is an area of concerted effort. Efficient nuclear entry is dependent on binding of the transcription factor MEF2, as mutations in the MEF2 binding region result in cytoplasmic accumulation of HDAC4. It is well established that nuclear exit and cytoplasmic retention are dependent on 14-3-3-binding, and mutations that affect binding are widely used to induce nuclear accumulation of HDAC4. While regulation of HDAC4 shuttling is clearly important, there is a gap in understanding of how the nuclear and cytoplasmic distribution of HDAC4 impacts its function. Furthermore, it is unclear whether other features of the protein including the catalytic site, the MEF2-binding region and/or the ankyrin repeat binding motif influence the distribution and/or activity of HDAC4 in neurons. Since HDAC4 functions are conserved in Drosophila, and increased nuclear accumulation of HDAC4 also results in impaired neurodevelopment, we used Drosophila as a genetic model for investigation of HDAC4 function. RESULTS: Here we have generated a series of mutants for functional dissection of HDAC4 via in-depth examination of the resulting subcellular distribution and nuclear aggregation, and correlate these with developmental phenotypes resulting from their expression in well-established models of neuronal morphogenesis of the Drosophila mushroom body and eye. We found that in the mushroom body, forced sequestration of HDAC4 in the nucleus or the cytoplasm resulted in defects in axon morphogenesis. The actions of HDAC4 that resulted in impaired development were dependent on the MEF2 binding region, modulated by the ankyrin repeat binding motif, and largely independent of an intact catalytic site. In contrast, disruption to eye development was largely independent of MEF2 binding but mutation of the catalytic site significantly reduced the phenotype, indicating that HDAC4 acts in a neuronal-subtype-specific manner. CONCLUSIONS: We found that the impairments to mushroom body and eye development resulting from nuclear accumulation of HDAC4 were exacerbated by mutation of the ankyrin repeat binding motif, whereas there was a differing requirement for the MEF2 binding site and an intact catalytic site. It will be of importance to determine the binding partners of HDAC4 in nuclear aggregates and in the cytoplasm of these tissues to further understand its mechanisms of action.
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    Ankyrin2 is essential for neuronal morphogenesis and long-term courtship memory in Drosophila
    (BioMed Central Ltd, 2023-05-16) Schwartz S; Wilson SJ; Hale TK; Fitzsimons HL
    Dysregulation of HDAC4 expression and/or nucleocytoplasmic shuttling results in impaired neuronal morphogenesis and long-term memory in Drosophila melanogaster. A recent genetic screen for genes that interact in the same molecular pathway as HDAC4 identified the cytoskeletal adapter Ankyrin2 (Ank2). Here we sought to investigate the role of Ank2 in neuronal morphogenesis, learning and memory. We found that Ank2 is expressed widely throughout the Drosophila brain where it localizes predominantly to axon tracts. Pan-neuronal knockdown of Ank2 in the mushroom body, a region critical for memory formation, resulted in defects in axon morphogenesis. Similarly, reduction of Ank2 in lobular plate tangential neurons of the optic lobe disrupted dendritic branching and arborization. Conditional knockdown of Ank2 in the mushroom body of adult Drosophila significantly impaired long-term memory (LTM) of courtship suppression, and its expression was essential in the γ neurons of the mushroom body for normal LTM. In summary, we provide the first characterization of the expression pattern of Ank2 in the adult Drosophila brain and demonstrate that Ank2 is critical for morphogenesis of the mushroom body and for the molecular processes required in the adult brain for the formation of long-term memories.
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    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
    (Massey University, 2022) Tan, Wei Jun
    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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    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
    (Massey University, 2019) Main, Patrick James
    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.
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    Investigating the role of histone deacetylase HDAC4 in long-term memory formation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Genetics at Massey University, Manawatu, New Zealand
    (Massey University, 2016) Schwartz, Silvia
    Epigenetic mechanisms are emerging as master regulators of cognitive abilities such as learning and memory. It has been previously shown that the histone deacetylase HDAC4 plays a critical role in memory formation in both mammals and insects although the specific mechanisms through which it acts have not yet been elucidated. HDAC4 undergoes nucleocytoplasmic shuttling and, in neurons, it is largely cytoplasmic implying it may play both nuclear and non-nuclear functions. To identify upstream regulators and downstream targets of HDAC4, a genetic interaction screen was performed in the fruit fly Drosophila melanogaster, a powerful model system to study the genetic mechanisms of neurological disease. Twenty-nine genes were found to interact with HDAC4 suggesting they are part of the same molecular pathway. Functional network analysis revealed that many of the genes could be grouped into three biological categories comprising transcriptional factors, SUMOylation machinery enzymes and cytoskeletal regulators/interactors. Within the latter, Ankyrin2 was selected for further analysis as it is implicated in synaptic stability and in human intellectual disability. In addition HDAC4 harbours a conserved ankyrin binding domain. Immunohistochemical analyses showed widespread distribution of Ankyrin2 throughout the adult brain and coincident distribution with HDAC4 was observed in the axons of the mushroom body, a key structure for memory formation in flies. Both HDAC4 and Ankyrin2 were also found to regulate mushroom body development. RNAi-mediated depletion of Ankyrin2 in the adult brain impaired long-term memory in the courtship suppression assay, a model of associative memory and preliminary evidence of a physical association between HDAC4 and Ankyrin2 was also demonstrated. The genes identified in the screen provide new avenues for investigation of the mechanisms through which HDAC4 regulates memory formation and preliminary analyses suggest that interaction with the cytoskeletal adaptor Ankyrin2 may involve remodelling of the actin/spectrin cytoskeleton, phenomenon that underlies memory related processes like synaptic plasticity and neuronal excitability.