Journal Articles
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Item N-terminal oligomerization drives HDAC4 nuclear condensation and neurodevelopmental dysfunction in Drosophila(The Royal Society, 2025-10) Hawley HR; Sutherland-Smith AJ; Savoian MS; Fitzsimons HLHistone deacetylase four (HDAC4) undergoes dynamic nucleocytoplasmic shuttling, a process critical for regulating its activity. However, aberrant nuclear accumulation of HDAC4 is associated with both neurodevelopmental and neurodegenerative disease, and in our Drosophila model, impairs normal neuronal development. Upon nuclear accumulation, HDAC4 forms biomolecular condensates, previously termed aggregates, that correlate with the severity of defects in development of the Drosophila mushroom body and adult eye. Here we determined that nuclear condensation of HDAC4 is dependent on self-oligomerization, and that impairing oligomerization reduces condensation and the severity of neurodevelopmental phenotypes in Drosophila. HDAC4 condensates are highly dynamic and are stabilized by the presence of MEF2, which promotes their formation, ultimately exacerbating phenotypic severity. These data provide insight into the role of HDAC4 condensates in normal neuronal function and suggest that their dysregulation may contribute to neurodevelopmental disorders. Consequently, targeting oligomerization of HDAC4 and its interaction with MEF2 present potential therapeutic strategies for diseases associated with HDAC4 nuclear accumulation.Item Oxalate-Degrading Bacillus subtilis Mitigates Urolithiasis in a Drosophila melanogaster Model.(American Society for Microbiology, 2020-10-01) Al KF; Daisley BA; Chanyi RM; Bjazevic J; Razvi H; Reid G; Burton JPKidney stones affect nearly 10% of the population in North America and are associated with high morbidity and recurrence, yet novel prevention strategies are lacking. Recent evidence suggests that the human gut microbiota can influence the development of nephrolithiasis, although clinical trials have been limited and inconclusive in determining the potential for microbially based interventions. Here, we used an established Drosophila melanogaster model of urolithiasis as a high-throughput screening platform for evaluation of the therapeutic potential of oxalate-degrading bacteria in calcium oxalate (CaOx) nephrolithiasis. The results demonstrated that Bacillus subtilis 168 (BS168) is a promising candidate based on its preferential growth in high oxalate concentrations, its ability to stably colonize the D. melanogaster intestinal tract for as long as 5 days, and its prevention of oxalate-induced microbiota dysbiosis. Single-dose BS168 supplementation exerted beneficial effects on D. melanogaster for as long as 14 days, decreasing stone burden in dissected Malpighian tubules and fecal excreta while increasing survival and behavioral markers of health over those of nonsupplemented lithogenic controls. These findings were complemented by in vitro experiments using the established MDCK renal cell line, which demonstrated that BS168 pretreatment prevented increased CaOx crystal adhesion and aggregation. Taking our results together, this study supports the notion that BS168 can functionally reduce CaOx stone burden in vivo through its capacity for oxalate degradation. Given the favorable safety profile of many B. subtilis strains already used as digestive aids and in fermented foods, these findings suggest that BS168 could represent a novel therapeutic adjunct to reduce the incidence of recurrent CaOx nephrolithiasis in high-risk patients.IMPORTANCE Kidney stone disease is a morbid condition that is increasing in prevalence, with few nonsurgical treatment options. The majority of stones are composed of calcium oxalate. Unlike humans, some microbes can break down oxalate, suggesting that microbial therapeutics may provide a novel treatment for kidney stone patients. This study demonstrated that Bacillus subtilis 168 (BS168) decreased stone burden, improved health, and complemented the microbiota in a Drosophila melanogaster urolithiasis model, while not exacerbating calcium oxalate aggregation or adhesion to renal cells in vitro These results identify this bacterium as a candidate for ameliorating stone formation; given that other strains of B. subtilis are components of fermented foods and are used as probiotics for digestive health, strain 168 warrants testing in humans. With the severe burden that recurrent kidney stone disease imposes on patients and the health care system, this microbial therapeutic approach could provide an inexpensive therapeutic adjunct.Item 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 HLDysregulation 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.Item 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 HLBACKGROUND: 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.Item 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 HLDysregulation 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.Item Increased Abundance of Nuclear HDAC4 Impairs Neuronal Development and Long-Term Memory(Frontiers Media, 30/03/2021) Fitzsimons H; Main P; Wheeler D; Tan WJDysregulation of the histone deacetylase HDAC4 is associated with both neurodevelopmental and neurodegenerative disorders, and a feature common to many of these disorders is impaired cognitive function. HDAC4 shuttles between the nucleus and cytoplasm in both vertebrates and invertebrates and alterations in the amounts of nuclear and/or cytoplasmic HDAC4 have been implicated in these diseases. In Drosophila, HDAC4 also plays a critical role in the regulation of memory, however, the mechanisms through which it acts are unknown. Nuclear and cytoplasmically-restricted HDAC4 mutants were expressed in the Drosophila brain to investigate a mechanistic link between HDAC4 subcellular distribution, transcriptional changes and neuronal dysfunction. Deficits in mushroom body morphogenesis, eye development and long-term memory correlated with increased abundance of nuclear HDAC4 but were associated with minimal transcriptional changes. Although HDAC4 sequesters MEF2 into punctate foci within neuronal nuclei, no alteration in MEF2 activity was observed on overexpression of HDAC4, and knockdown of MEF2 had no impact on long-term memory, indicating that HDAC4 is likely not acting through MEF2. In support of this, mutation of the MEF2 binding site within HDAC4 also had no impact on nuclear HDAC4-induced impairments in long-term memory or eye development. In contrast, the defects in mushroom body morphogenesis were ameliorated by mutation of the MEF2 binding site, as well as by co-expression of MEF2 RNAi, thus nuclear HDAC4 acts through MEF2 to disrupt mushroom body development. These data provide insight into the mechanisms through which dysregulation of HDAC4 subcellular distribution impairs neurological function and provides new avenues for further investigation.Item Loss of Drosophila Coq8 results in impaired survival, locomotor deficits and photoreceptor degeneration(BioMed Central, 9/02/2022) Hura A; Hannah H; Tan WJ; Penny R; Jessie J; Fitzsimons HCoenzyme Q8A encodes the homologue of yeast coq8, an ATPase that is required for the biosynthesis of Coenzyme Q10, an essential component of the electron transport chain. Mutations in COQ8A in humans result in CoQ10 deficiency, the clinical features of which include early-onset cerebellar ataxia, seizures and intellectual disability. The rapid advancement of massively parallel sequencing has resulted in the identification of more than 40 new mutations in COQ8A and functional studies are required to confirm causality and to further research into determining the specific mechanisms through which the mutations result in loss of function. To that end, a Drosophila model of Coq8 deficiency was developed and characterized to determine its appropriateness as a model system to further explore the role of Coq8 in the brain, and for functional characterisation of Coq8 mutations. Pan-neuronal RNAi knockdown of Coq8 was largely lethal, with female escapers displaying severe locomotor deficits. Knockdown of Coq8 in the eye resulted in degeneration of photoreceptors, progressive necrosis and increased generation of reactive oxygen species. Reintroduction of wild-type Coq8 restored normal function, however expression of human wild-type COQ8A exacerbated the eye phenotype, suggesting it was acting as a dominant-negative. This model is therefore informative for investigating the function of Drosophila Coq8, however human COQ8A mutations cannot be assessed as hCOQ8A does not rescue Coq8 deficiency.
