Now showing 1 - 4 of 4
  • Publication
    A method for isolation of cone photoreceptors from adult zebrafish retinae
    Background: Cone photoreceptors are specialised sensory retinal neurons responsible for photopic vision, colour perception and visual acuity. Retinal degenerative diseases are a heterogeneous group of eye diseases in which the most severe vision loss typically arises from cone photoreceptor dysfunction or degeneration. Establishing a method to purify cone photoreceptors from retinal tissue can accelerate the identification of key molecular determinants that underlie cone photoreceptor development, survival and function. The work herein describes a new method to purify enhanced green fluorescent protein (EGFP)-labelled cone photoreceptors from adult retina of Tg(3.2gnat2:EGFP) zebrafish. Results: Methods for dissecting adult zebrafish retinae, cell dissociation, cell sorting, RNA isolation and RNA quality control were optimised. The dissociation protocol, carried out with ~30 retinae from adult zebrafish, yielded approximately 6 × 106 cells. Flow cytometry cell sorting subsequently distinguished 1 × 106 EGFP+ cells and 4 × 106 EGFP− cells. Electropherograms confirmed downstream isolation of high-quality RNA with RNA integrity number (RIN) >7.6 and RNA concentration >5.7 ng/µl obtained from both populations. Reverse Transcriptase-PCR confirmed that the EGFP-positive cell populations express known genetic markers of cone photoreceptors that were not expressed in the EGFP-negative cell population whereas a rod opsin amplicon was only detected in the EGFP-negative retinal cell population. Conclusions: This work describes a valuable adult zebrafish cone photoreceptor isolation methodology enabling future identification of cone photoreceptor-enriched genes, proteins and signalling networks responsible for their development, survival and function. In addition, this advancement facilitates the identification of novel candidate genes for inherited human blindness.
      627Scopus© Citations 6
  • Publication
    GATD3A, a mitochondrial deglycase with evolutionary origins from gammaproteobacteria, restricts the formation of advanced glycation end products
    Background: Functional complexity of the eukaryotic mitochondrial proteome is augmented by independent gene acquisition from bacteria since its endosymbiotic origins. Mammalian homologs of many ancestral mitochondrial proteins have uncharacterized catalytic activities. Recent forward genetic approaches attributed functions to proteins in established metabolic pathways, thereby limiting the possibility of identifying novel biology relevant to human disease. We undertook a bottom-up biochemistry approach to discern evolutionarily conserved mitochondrial proteins with catalytic potential. Results: Here, we identify a Parkinson-associated DJ-1/PARK7-like protein—glutamine amidotransferase-like class 1 domain-containing 3A (GATD3A), with bacterial evolutionary affinities although not from alphaproteobacteria. We demonstrate that GATD3A localizes to the mitochondrial matrix and functions as a deglycase. Through its amidolysis domain, GATD3A removes non-enzymatic chemical modifications produced during the Maillard reaction between dicarbonyls and amines of nucleotides and amino acids. GATD3A interacts with factors involved in mitochondrial mRNA processing and translation, suggestive of a role in maintaining integrity of important biomolecules through its deglycase activity. The loss of GATD3A in mice is associated with accumulation of advanced glycation end products (AGEs) and altered mitochondrial dynamics. Conclusions: An evolutionary perspective helped us prioritize a previously uncharacterized but predicted mitochondrial protein GATD3A, which mediates the removal of early glycation intermediates. GATD3A restricts the formation of AGEs in mitochondria and is a relevant target for diseases where AGE deposition is a pathological hallmark.
      100Scopus© Citations 5
  • Publication
    Phenotype-based Discovery of 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol as a Novel Regulator of Ocular Angiogenesis
    Retinal angiogenesis is tightly regulated to meet oxygenation and nutritional requirements. In diseases such as proliferative diabetic retinopathy and neovascular age-related macular degeneration, uncontrolled angiogenesis can lead to blindness. Our goal is to better understand the molecular processes controlling retinal angiogenesis and discover novel drugs that inhibit retinal neovascularization. Phenotype-based chemical screens were performed using the ChemBridge DiversetTM library and inhibition of hyaloid vessel angiogenesis in Tg(fli1:EGFP) zebrafish. 2-[(E)-2-(Quinolin-2-yl)vinyl]phenol, (quininib) robustly inhibits developmental angiogenesis at 4–10 μm in zebrafish and significantly inhibits angiogenic tubule formation in HMEC-1 cells, angiogenic sprouting in aortic ring explants, and retinal revascularization in oxygen-induced retinopathy mice. Quininib is well tolerated in zebrafish, human cell lines, and murine eyes. Profiling screens of 153 angiogenic and inflammatory targets revealed that quininib does not directly target VEGF receptors but antagonizes cysteinyl leukotriene receptors 1 and 2 (CysLT1–2) at micromolar IC50 values. In summary, quininib is a novel anti-angiogenic small-molecule CysLT receptor antagonist. Quininib inhibits angiogenesis in a range of cell and tissue systems, revealing novel physiological roles for CysLT signaling. Quininib has potential as a novel therapeutic agent to treat ocular neovascular pathologies and may complement current anti-VEGF biological agents.
      515Scopus© Citations 32
  • Publication
    Genome editing: the breakthrough technology for inherited retinal disease?
    Introduction: Genetic alterations resulting in a dysfunctional retinal pigment epithelium and/or degenerating photoreceptors cause impaired vision. These juxtaposed cells in the retina of the posterior eye are crucial for the visual cycle or phototransduction. Deficits in these biochemical processes perturb neural processing of images capturing the external environment. Notably, there is a distinct lack of clinically approved pharmacological, cell- or gene-based therapies for inherited retinal disease. Gene editing technologies are rapidly advancing as a realistic therapeutic option. Areas Covered: Recent discovery of endonuclease-mediated gene editing technologies has culminated in a surge of investigations into their therapeutic potential. In this review, the authors discuss gene editing technologies and their applicability in treating inherited retinal diseases, the limitations of the technology and the research obstacles to overcome before editing a patient’s genome becomes a viable treatment option. Expert Opinion: The ability to strategically edit a patient’s genome constitutes a treatment revolution. However, concerns remain over the safety and efficacy of either transplanting iPSC-derived retinal cells following ex vivo gene editing, or with direct gene editing in vivo. Ultimately, further refinements to improve efficacy and safety profiles are paramount for gene editing to emerge as a widely available treatment option.
      746Scopus© Citations 9