Browsing by Author "Fitzsimons H"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    Combined injection of rAAV with mannitol enhances gene expression in the rat brain
    (Cell Press, 6/09/2000) Mastakov M; Baer K; Xu R; Fitzsimons H; During M
    Recombinant adeno-associated viruses (rAAV) are highly efficient vectors for gene transfer into the central nervous system (CNS). However, a major hurdle for gene delivery to the mammalian brain is to achieve high-level transduction in target cells beyond the immediate injection site. Therefore, in addition to improvements in expression cassettes and viral titers, optimal injection parameters need to be defined. Here, we show that previous studies of somatic cell gene transfer to the mammalian brain have used suboptimal injection parameters, with even the lowest reported perfusion rates still excessively fast. Moreover, we evaluated the effect of local administration of mannitol to further enhance transgene expression and vector spread. Ultraslow microperfusion of rAAV, i.e., <33 nl/min, resulted in significantly higher gene expression and less injury of surrounding tissue than the previously reported rates of 100 nl/min or faster. Co-infusion of mannitol facilitated gene transfer to neurons, increasing both the total number and the distribution of transduced cells by 200-300%. Gene transfer studies in the CNS using rAAV should use very slow infusion rates and combined injection with mannitol to maximize transduction efficiency and spread.
  • Thumbnail Image
    Item
    Herzog is not required for mushroom body development or courtship learning & memory but is required for eye development in Drosophila melanogaster
    (30/01/2023) Fitzsimons H; Palmer MJ
    Herzog (Hzg, CG5830) shares similarity to members of the haloacid dehalogenase subfamily of small CTD phosphatases. In Drosophila it is a maternal gene essential for establishment of embryonic segment polarity, and oligomerization is required for activation of phosphatase activity. While Hzg is expressed in the brain, its role has not been investigated. To that end, we further characterized Hzg expression in the brain and found that it is highly expressed in neurons of the mushroom body where it localises to axons, and is also expressed in cortical glia. We investigated its role in mushroom body development as well as courtship learning and memory, but found that knockdown of Hzg had no impact on these processes. In contrast, knockdown in post-mitotic neurons in the eye resulted in disruption to ommatidial patterning and pigmentation, indicating it plays an important role in eye development.
  • Thumbnail Image
    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 WJ
    Dysregulation 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.
  • Thumbnail Image
    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 H
    Coenzyme 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.