James Reilly

A colour preference technique to evaluate acrylamide-induced toxicity in zebrafish

The zebrafish has become a commonly used vertebrate model for toxicity assessment, of particular relevance to the study of toxic effects on the visual system because of the structural similarities shared by zebrafish and human retinae. In this article we present a colour preference-based technique that, by assessing the functionality of photoreceptors, can be used to evaluate the effects of toxicity on behaviour. A digital camera was used to record the locomotor behaviour of individual zebrafish swimming in a water tank consisting of two compartments separated by an opaque perforated wall through which the fish could pass. The colour of the lighting in each compartment could be altered independently (producing distinct but connected environments of white, red or blue) to allow association of the zebrafishs swimming behaviour with its colour preference. The functionality of the photoreceptors was evaluated based on the ability of the zebrafish to sense the different colours and to swim between the compartments. The zebrafish tracking was carried out using our algorithm developed with MATLAB. We found that zebrafish preferred blue illumination to white, and white illumination to red. Acute treatment with acrylamide (2mM for 36h) resulted in a marked reduction in locomotion and a concomitant loss of colour-preferential swimming behaviour. Histopathological examination of acrylamide-treated zebrafish eyes showed that acrylamide exposure had caused retinal damage. The colour preference tracking technique has applications in the assessment of neurodegenerative disorders, as a method for preclinical appraisal of drug efficacy and for behavioural evaluation of toxicity.

Therapeutic Potential of Co-enzyme Q10 in Retinal Diseases

BACKGROUND Coenzyme Q10 (CoQ10) plays a critical role in mitochondrial oxidative phosphorylation by serving as an electron carrier in the respiratory electron transport chain. CoQ10 also functions as a lipid-soluble antioxidant by protecting lipids, proteins and DNA damaged by oxidative stress. CoQ10 deficiency has been associated with a number of human diseases in which CoQ10 supplementation therapy has been effective in slowing or reversing pathological changes. Oxidative stress is a major contributory factor in the process of retinal degeneration. METHOD The related literature was reviewed through searching PubMed using keywords: CoQ10, CoQ10 and oxidative stress, CoQ10 and retinal degeneration. The functions of CoQ10 were summarized and its use in the treatment of age-related macular degeneration and glaucoma highlighted. The therapeutic potential of CoQ10 for other retinal diseases was also discussed. RESULTS CoQ10 has been applied in different types of neurodegeneration. CoQ10 is detectable in retina and declines with ageing. Early studies showed treatment of CoQ10 improved visual function in patients with age-related macular degeneration. In glaucomatous models, CoQ10 exposure protected ganglion cell death from environmental stress; in glaucoma patients, CoQ10 treatment demonstrated beneficial effects on function of inner retina and enhancement of visual cortical response. Since oxidative stress also plays a critical role in the pathogenesis of diabetic retinopathy and retinitis pigmentosa, CoQ10 is a therapeutic target for both conditions. CONCLUSION A wide range of evidence supports a role of CoQ10 in retinal diseases through inhibiting production of reactive oxygen species and protecting neuroretinal cells from oxidative damage.

CERKL gene knockout disturbs photoreceptor outer segment phagocytosis and causes rod-cone dystrophy in zebrafish

In humans, CERKL mutations cause widespread retinal degeneration: early dysfunction and loss of rod and cone photoreceptors in the outer retina and, progressively, death of cells in the inner retina. Despite intensive efforts, the function of CERKL remains obscure and studies in animal models have failed to clarify the disease mechanism of CERKL mutations. To address this gap in knowledge, we have generated a stable CERKL knockout zebrafish model by TALEN technology and a 7bp deletion in CERKL cDNA that caused the premature termination of CERKL. These CERKL-/- animals showed progressive degeneration of photoreceptor outer segments (OSs) and increased apoptosis of retinal cells, including those in the outer and inner retinal layers. Additionally, we confirmed by immunofluorescence and western-blot that rod degeneration in CERKL-/- zebrafish occurred earlier and was more significant than that in cone cells. Accumulation of shed OSs in the interphotoreceptor matrix was observed by transmission election microscopy (TEM). This suggested that CERKL may regulate the phagocytosis of OSs by the retinal pigment epithelium (RPE). We further found that the phagocytosis-associated protein MERTK was significantly reduced in CERKL-/- zebrafish. Additionally, in ARPE-19 cell lines, knockdown of CERKL also decreased the mRNA and protein level of MERTK, as well as the ox-POS phagocytosis. We conclude that CERKL deficiency in zebrafish may cause rod-cone dystrophy, but not cone-rod dystrophy, while interfering with the phagocytosis function of RPE associated with down-regulation of the expression of MERTK.

Gypenosides protect retinal pigment epithelium cells from oxidative stress

Oxidative stress plays a critical role in the pathogenesis of retinal degeneration. Gypenosides are the major functional components isolated from Gynostemma pentaphyllum. They have been shown to protect against oxidative stress and inflammation and have also demonstrated a protective effect on experimental optic neuritis. In order to determine the protective properties of gypenosides against oxidative stress in human retinal pigment epithelium (RPE) cells, ARPE-19 cells were treated with H2O2 or H2O2 plus gypenosides for 24 h. ARPE-19 cells co-treated with gypenosides had significantly increased cell viability and decreased cell death rate when compared to cells treated with H2O2 alone. The level of GSH, the activities of SOD and catalase, and the expression of NRF2 and antioxidant genes were notably decreased, while there were marked increases in ROS, MDA and pro-inflammatory cytokines in ARPE-19 cells exposed to H2O2; co-treatment with gypenosides significantly counteract these changes. Our study suggests that gypenosides protect RPE cells from oxidative damage and offer therapeutic potential for the treatment of retinal degeneration.

Abnormal photoreceptor outer segment development and early retinal degeneration in kif3a mutant zebrafish

Photoreceptors are highly specialized sensory neurons that possess a modified primary cilium called the outer segment. Photoreceptor outer segment formation and maintenance require highly active protein transport via a process known as intraflagellar transport. Anterograde transport in outer segments is powered by the heterotrimeric kinesin II and coordinated by intraflagellar transport proteins. Here, we describe a new zebrafish model carrying a nonsense mutation in the kinesin II family member 3A (kif3a) gene. Kif3a mutant zebrafish exhibited curved body axes and kidney cysts. Outer segments were not formed in most parts of the mutant retina, and rhodopsin was mislocalized, suggesting KIF3A has a role in rhodopsin trafficking. Both rod and cone photoreceptors degenerated rapidly between 4 and 9 days post fertilization, and electroretinography response was not detected in 7 days post fertilization mutant larvae. Loss of KIF3A in zebrafish also resulted in an intracellular transport defect affecting anterograde but not retrograde transport of organelles. Our results indicate KIF3A plays a conserved role in photoreceptor outer segment formation and intracellular transport.

RPGR protein complex regulates proteasome activity and mediates store-operated calcium entry

Ciliopathies are a group of genetically heterogeneous disorders, characterized by defects in cilia genesis or maintenance. Mutations in the RPGR gene and its interacting partners, RPGRIP1 and RPGRIP1L, cause ciliopathies, but the function of their proteins remains unclear. Here we show that knockdown (KD) of RPGR, RPGRIP1 or RPGRIP1L in hTERT-RPE1 cells results in abnormal actin cytoskeleton organization. The actin cytoskeleton rearrangement is regulated by the small GTPase RhoA via the planar cell polarity (PCP) pathway. RhoA activity was upregulated in the absence of RPGR, RPGRIP1 or RPGRIP1L proteins. In RPGR, RPGRIP1 or RPGRIP1L KD cells, we observed increased levels of DVl2 and DVl3 proteins, the core components of the PCP pathway, due to impaired proteasomal activity. RPGR, RPGRIP1 or RPGRIP1L KD cells treated with thapsigargin (TG), an inhibitor of sarcoendoplasmic reticulum Ca2+- ATPases, showed impaired store-operated Ca2+ entry (SOCE), which is mediated by STIM1 and Orai1 proteins. STIM1 was not localized to the ER-PM junction upon ER store depletion in RPGR, RPGRIP1 or RPGRIP1L KD cells. Our results demonstrate that the RPGR protein complex is required for regulating proteasomal activity and for modulating SOCE, which may contribute to the ciliopathy phenotype.

Comparative analysis of the toxicity of gold nanoparticles in zebrafish

The use of nanoparticles – particles that range in size from 1 to 100 nm – has become increasingly prevalent in recent years, bringing with it a variety of potential toxic effects. Zebrafish embryos were exposed during the 3 day postfertilization period to gold nanospheres (GNSs), gold nanorods (GNRs), GNRs coated with polystyrene sulphate (PSS‐GNRs) and GNRs coated with both PSS and polyallamine hydrochloride (PAH‐PSS‐GNRs). All nanorods were stabilized with cetyltrimethylammonium bromide. GNSs were the least toxic of the nanoparticles studied, with exposure resulting in no significant changes in mortality, hatching or heart rate. Exposure to GNRs and PSS‐GNRs resulted in significant increases in mortality and significant decreases in hatching and heart rate. Treatment with GNRs caused significant changes in the expression of a variety of oxidative stress genes. The toxic effects of GNRs were ameliorated by coating them with PSS and, to a more marked extent, with a double coating of PSS and polyallamine hydrochloride.

Carnosic acid attenuates acrylamide-induced retinal toxicity in zebrafish embryos

Abstract Acrylamide (ACR) is a water‐soluble chemical used widely in industry, which can be formed in tobacco smoke and in starchy foods cooked at high temperatures. ACR is considered to be a neurotoxin, genotoxin and carcinotoxin. Previous studies reported that ACR‐exposed workers and experimental animals exhibited visual function defects, although the underlying mechanisms have not been elucidated. In this study, we found that zebrafish embryos exposed to 1 mM and 2 mM ACR showed significantly increased reactive oxygen species (ROS), decreased expression of the antioxidant genes Sod1, Sod2, Catalase, Gpx1 and Nrf2, reduced activity of superoxide dismutase (SOD) and catalase, and elevated malondialdehyde (MDA), compared with control embryos. ACR exposure caused loss of both rod and cone photoreceptor cells through Caspase‐3‐dependent apoptotis. When embryos were simultaneously exposed to ACR and the natural antioxidative substance carnosic acid (CA), the presence of the latter (10 &mgr;M) markedly counteracted the above ACR‐induced toxic effects. Our data suggest that CA can protect photoreceptor cells against ACR‐induced oxidative damage and has a potential for neuroprotection of visual function in humans exposed to ACR. HighlightsAcrylamide induced overproduction of ROS in zebrafish embryos.Acrylamide exposure resulted in apoptotic death of photoreceptor cells in zebrafish embryos.Carnosic acid attenuated acrylamide‐induced retinal toxicity in zebrafish embryos.

CERKL regulates autophagy via the NAD-dependent deacetylase SIRT1

ABSTRACT Macroautophagy/autophagy is an important intracellular mechanism for the maintenance of cellular homeostasis. Here we show that the CERKL (ceramide kinase like) gene, a retinal degeneration (RD) pathogenic gene, plays a critical role in regulating autophagy by stabilizing SIRT1. In vitro and in vivo, suppressing CERKL results in impaired autophagy. SIRT1 is one of the main regulators of acetylation/deacetylation in autophagy. In CERKL-depleted retinas and cells, SIRT1 is downregulated. ATG5 and ATG7, 2 essential components of autophagy, show a higher degree of acetylation in CERKL-depleted cells. Overexpression of SIRT1 rescues autophagy in CERKL-depleted cells, whereas CERKL loses its function of regulating autophagy in SIRT1-depleted cells, and overexpression of CERKL upregulates SIRT1. Finally, we show that CERKL directly interacts with SIRT1, and may regulate its phosphorylation at Ser27 to stabilize SIRT1. These results show that CERKL is an important regulator of autophagy and it plays this role by stabilizing the deacetylase SIRT1.