The splicing factor Prpf31 is required for hematopoietic stem and progenitor cell expansion during zebrafish embryogenesis.

Pre-mRNA splicing is a precise regulated process and is crucial for system development and homeostasis maintenance. Mutations in spliceosomal components have been found in various hematopoietic malignancies (HMs) and have been considered as oncogenic derivers of HMs. However, the role of spliceosomal components in normal and malignant hematopoiesis remains largely unknown. Pre-mRNA processing factor 31 (PRPF31) is a constitutive spliceosomal component, which mutations are associated with autosomal dominant retinitis pigmentosa. PRPF31 was found to be mutated in several HMs, but the function of PRPF31 in normal hematopoiesis has not been explored. In our previous study, we generated a prpf31 knockout (KO) zebrafish line and reported that Prpf31 regulates the survival and differentiation of retinal progenitor cells by modulating the alternative splicing of genes involved in mitosis and DNA repair. In this study, by using the prpf31 KO zebrafish line, we discovered that prpf31 KO zebrafish exhibited severe defects in hematopoietic stem and progenitor cell (HSPC) expansion and its sequentially differentiated lineages. Immunofluorescence results showed that Prpf31-deficient HSPCs underwent malformed mitosis and M phase arrest during HSPC expansion. Transcriptome analysis and experimental validations revealed that Prpf31 deficiency extensively perturbed the alternative splicing of mitosis-related genes. Collectively, our findings elucidate a previously undescribed role for Prpf31 in HSPC expansion, through regulating the alternative splicing of mitosis-related genes.

Rod genesis driven by mafba in an nrl knockout zebrafish model with altered photoreceptor composition and progressive retinal degeneration.

Neural retina leucine zipper (NRL) is an essential gene for the fate determination and differentiation of the precursor cells into rod photoreceptors in mammals. Mutations in NRL are associated with the autosomal recessive enhanced S-cone syndrome and autosomal dominant retinitis pigmentosa. However, the exact role of Nrl in regulating the development and maintenance of photoreceptors in the zebrafish (Danio rerio), a popular animal model used for retinal degeneration and regeneration studies, has not been fully determined. In this study, we generated an nrl knockout zebrafish model via the CRISPR-Cas9 technology and observed a surprising phenotype characterized by a reduced number, but not the total loss, of rods and over-growth of green cones. We discovered two waves of rod genesis, nrl-dependent and -independent at the embryonic and post-embryonic stages, respectively, in zebrafish by monitoring the rod development. Through bulk and single-cell RNA sequencing, we characterized the gene expression profiles of the whole retina and each retinal cell type from the wild type and nrl knockout zebrafish. The over-growth of green cones and mis-expression of green-cone-specific genes in rods in nrl mutants suggested that there are rod/green-cone bipotent precursors, whose fate choice between rod versus green-cone is controlled by nrl. Besides, we identified the mafba gene as a novel regulator of the nrl-independent rod development, based on the cell-type-specific expression patterns and the retinal phenotype of nrl/mafba double-knockout zebrafish. Gene collinearity analysis revealed the evolutionary origin of mafba and suggested that the function of mafba in rod development is specific to modern fishes. Furthermore, the altered photoreceptor composition and abnormal gene expression in nrl mutants caused progressive retinal degeneration and subsequent regeneration. Accordingly, this study revealed a novel function of the mafba gene in rod development and established a working model for the developmental and regulatory mechanisms regarding the rod and green-cone photoreceptors in zebrafish.

Prpf31 is essential for the survival and differentiation of retinal progenitor cells by modulating alternative splicing.

Dysfunction of splicing factors often result in abnormal cell differentiation and apoptosis, especially in neural tissues. Mutations in pre-mRNAs processing factor 31 (PRPF31) cause autosomal dominant retinitis pigmentosa, a progressive retinal degeneration disease. The transcriptome-wide splicing events specifically regulated by PRPF31 and their biological roles in the development and maintenance of retina are still unclear. Here, we showed that the differentiation and viability of retinal progenitor cells (RPCs) are severely perturbed in prpf31 knockout zebrafish when compared with other tissues at an early embryonic stage. At the cellular level, significant mitotic arrest and DNA damage were observed. These defects could be rescued by the wild-type human PRPF31 rather than the disease-associated mutants. Further bioinformatic analysis and experimental verification uncovered that Prpf31 deletion predominantly causes the skipping of exons with a weak 5' splicing site. Moreover, genes necessary for DNA repair and mitotic progression are most enriched among the differentially spliced events, which may explain the cellular and tissular defects in prpf31 mutant retinas. This is the first time that Prpf31 is demonstrated to be essential for the survival and differentiation of RPCs during retinal neurogenesis by specifically modulating the alternative splicing of genes involved in DNA repair and mitosis.

Urocanic acid enhances memory consolidation and reconsolidation in novel object recognition task.

Urocanic acid (UCA) is an endogenous small molecule that is elevated in skin, blood and brain after sunlight exposure, mainly playing roles in the periphery systems. Few studies have investigated the role of UCA in the central nervous system. In particular, its role in memory consolidation and reconsolidation is still unclear. In the present study, we investigated the effect of intraperitoneal injection of UCA on memory consolidation and reconsolidation in a novel object recognition memory (ORM) task. In the consolidation version of the ORM task, the protocol involved three phases: habituation, sampling and test. UCA injection immediately after the sampling period enhanced ORM memory performance; UCA injection 6 h after sampling did not affect ORM memory performance. In the reconsolidation version of the ORM task, the protocol involved three phases: sampling, reactivation and test. UCA injection immediately after reactivation enhanced ORM memory performance; UCA injection 6 h after reactivation did not affect ORM memory performance; UCA injection 24 h after sampling without reactivation did not affect ORM memory performance. This UCA-enhanced memory performance was not due to its effects on nonspecific responses such as locomotor activity and exploratory behavior. The results suggest that UCA injection enhances consolidation and reconsolidation of an ORM task, which further extends previous research on UCA effects on learning and memory.

Effect of a TSPO ligand on retinal pigment epithelial cholesterol homeostasis in high-fat fed mice, implication for age-related macular degeneration.

Age-related Macular Degeneration (AMD) is a major cause of sight impairment in the elderly with complex aetiology involving genetics and environment and with limited therapeutic options which have limited efficacy. We have previously shown in a mouse-model of the condition, induced by feeding a high fat diet, that adverse effects of the diet can be reversed by co-administration of the TSPO activator, etifoxine. We extend those observations showing improvements in retinal pigment epithelial (RPE) cells with decreased lipids and enhanced expression of cholesterol metabolism and transport enzymes. Further, etifoxine decreased levels of reactive oxygen species (ROS) in RPE and inflammatory cytokines in RPE and serum. With respect to gut microbiome, we found that organisms abundant in the high fat condition (e.g. in the genus Anaerotruncus and Oscillospira) and implicated in AMD, were much less abundant after etifoxine treatment. The changes in gut flora were associated with the predicted production of metabolites of benefit to the retina including tryptophan and other amino acids and taurine, an essential component of the retina necessary to counteract ROS. These novel observations strengthen earlier conclusions that the mechanisms behind improvements in etifoxine-induced retinal physiology involve an interaction between effects on the host and the gut microbiome.

The chromatin remodeler Brg1 is required for formation and maintenance of hematopoietic stem cells.

Hematopoietic stem and progenitor cells (HSPCs) have the ability to self-renew and differentiate into various blood cells, thus playing an important role in maintenance of lifelong hematopoiesis. Brahma-related gene 1 (BRG1), which acts as the ATP subunit of mammalian SWI-SNF-related chromatin remodeling complexes, is involved in human acute myeloid leukemia and highly expresses in short-term HSPCs. But its role and regulatory mechanism for HSPC development have not yet been well established. Here, we generated a brg1 knockout zebrafish model using TALEN technology. We found that in brg1-/- embryo, the primitive hematopoiesis remained well, while definitive hematopoiesis formation was significantly impaired. The number of hemogenic endothelial cells was decreased, further affecting definitive hematopoiesis with reduced myeloid and lymphoid cells. During embryogenesis, the nitric oxide (NO) microenvironment in brg1-/- embryo was seriously damaged and the reduction of HSPCs could be partially rescued by a NO donor. Chromatin immunoprecipitation (ChIP) assays showed that BRG1 could bind to the promoter of KLF2 and trigger its transcriptional activity of NO synthase. Our findings show that Brg1 promotes klf2a expression in hemogenic endothelium and highlight a novel mechanism for HSPC formation and maintenance.

Protection by vitamin D against high-glucose-induced damage in retinal pigment epithelial cells.

Diabetic retinopathy (DR) is a diabetes-associated complication characterized by irreversible deterioration of the microvessels within the retina, leading subsequently to severe retinal damage and vision loss. Vitamin D (VITD), a steroid hormone, plays multiple physiological functions in cellular homeostasis. Deficiency of VITD has been suggested to be associated with DR. To study the potential protective function of VITD in DR, high-glucose-treated ARPE-19 cells and STZ-induced diabetic mice were used as in vitro and in vivo models. The protective effects of VITD were assessed based on the changes of expression of antioxidant enzymes and cytokines in high-glucose-treated retinal pigment epithelial (RPE) cells and in the retina and RPE of diabetic and VITD-treated diabetic mice. The present study demonstrated that exposure to a high level of glucose caused upregulation of pro-inflammatory cytokines and a decrease in anti-oxidant enzyme expression in both in vitro and in vivo models. VITD treatment increased cell viability, reduced reactive oxygen species (ROS) production and caspase-3/7 activities in high-glucose-treated RPE cells. Our data suggest that VITD can protect the retina and RPE from high-glucose-induced oxidative damage and inflammation.

Sub-clinical thickening of the fovea in diabetes and its relationship to glycaemic control: a study using swept-source optical coherence tomography.

BACKGROUND: Accumulation of multiple pockets of fluid at the fovea, as a complication of poor blood glucose control in diabetes, causes impairment of central vision. A new ability to demonstrate a pre-clinical phase of this maculopathy could be valuable, enabling diabetic individuals to be alerted to the need to improve their glycaemic control. This study aimed to use swept-source optical coherence tomography (SS-OCT) to measure foveal thickness and macular volume in diabetic individuals without cystoid macular oedema, and in non-diabetic individuals, and relate these measures to participants' glycaemic control. METHODS: Centre point thickness (CPT) and total macular volume (TMV) were measured using SS-OCT (DRI OCT Triton™, Topcon, Tokyo, Japan). Participants' glycosylated haemoglobin (HbA1c) level was also assessed (A1cNow®+ System, PTS Diagnostics, Indianapolis, IN, USA). The diabetic (n = 27) and non-diabetic (n = 27) groups were matched for age (p = 0.100) and sex (p = 0.414), and HbA1c level differed between diabetic and non-diabetic groups (p < 0.0005). The diabetic group comprised type 1 (n = 7) and type 2 (n = 20) diabetic individuals who were matched for duration of diabetes (p = 0.617) and whose glycaemic control was similar (p = 0.814). RESULTS: Diabetic individuals had significantly higher CPT (t(37) = 3.859, p < 0.0005) than non-diabetic individuals. In the diabetic group, multiple linear regression analysis revealed a conspicuous relationship between CPT and HbA1c level (ß = 0.501, t(21) = 3.139, p = 0.005): there was a 19-µm increase in CPT for each 1% increase in HbA1c level. This relationship was not present in the non-diabetic group (ß = - 0.068, t(23) = - 0.373, p = 0.712). CONCLUSIONS: SS-OCT is the only way to measure macular thickness in vivo. Diabetic individuals en bloc had higher CPT compared with non-diabetic individuals. Moreover, in the diabetic group, HbA1c level significantly predicted CPT. Our results suggest that, in diabetes, sub-clinical thickening may occur at the fovea before cystoid macular oedema becomes clinically evident. This could provide diabetic individuals with an early warning of disease progression and motivate them to improve control of their diabetes, with a view to avoiding the need of intra-vitreal injections with their attendant risks.

Deletion of the transmembrane protein Prom1b in zebrafish disrupts outer-segment morphogenesis and causes photoreceptor degeneration.

Mutations in human prominin 1 (PROM1), encoding a transmembrane glycoprotein localized mainly to plasma membrane protrusions, have been reported to cause retinitis pigmentosa, macular degeneration, and cone-rod dystrophy. Although the structural role of PROM1 in outer-segment (OS) morphogenesis has been demonstrated in Prom1-knockout mouse, the mechanisms underlying these complex disease phenotypes remain unclear. Here, we utilized a zebrafish model to further investigate PROM1's role in the retina. The Prom1 orthologs in zebrafish include prom1a and prom1b, and our results showed that prom1b, rather than prom1a, plays an important role in zebrafish photoreceptors. Loss of prom1b disrupted OS morphogenesis, with rods and cones exhibiting differences in impairment: cones degenerated at an early age, whereas rods remained viable but with an abnormal OS, even at 9 months postfertilization. Immunofluorescence experiments with WT zebrafish revealed that Prph2, an ortholog of the human transmembrane protein peripherin 2 and also associated with OS formation, is localized to the edge of OS and is more highly expressed in the cone OS than in the rod OS. Moreover, we found that Prom1b deletion causes mislocalization of Prph2 and disrupts its oligomerization. We conclude that the variation in Prph2 levels between cones and rods was one of the reasons for the different PROM1 mutation-induced phenotypes of these retinal structures. These findings expand our understanding of the phenotypes caused by PROM1 mutations and provide critical insights into its function.

Gypenosides mediate cholesterol efflux and suppress oxidized LDL induced inflammation in retinal pigment epithelium cells.

Age-related macular degeneration (AMD) is a predominant cause of visual deficit in aged population. Abnormal accumulation of cholesterol, including oxidized low-density lipoprotein (oxLDL), underneath the retinal pigment epithelium (RPE) cells contributes to the development of AMD. Gypenosides (Gyp) are glycosides extracted from Gynostemma pentaphyllum and have demonstrated protective effects against inflammation and oxidative stress. To determine the therapeutic potential of Gyp for AMD, we investigated its effect on cholesterol trafficking and metabolism and assessed the protective function of Gyp against oxLDL-induced damage in RPE cells. Cholesterol efflux to high-density lipoprotein (HDL) and human serum was significantly increased in RPE cells treated with Gyp when compared to untreated control cells. Expression of cholesterol metabolism (CYP27A1, CYP46A1) and trafficking (TSPO, ABCA1 and ABCG1) genes was also markedly increased in Gyp-treated RPE cells. OxLDL-treated RPE cells had significantly increased cholesterol accumulation and lipid droplet formation. There were marked increases in reactive oxygen species (ROS) generation and proinflammatory cytokines via NF-κB activation in RPE cells treated with oxLDL, while incubation with Gyp rectified these changes. These findings provide pharmacological evidence that Gyp has the potential to treat patients with early onset AMD by promoting cellular cholesterol removal from RPE cells and inhibiting inflammation and oxidative stress.

Gypenosides attenuate retinal degeneration in a zebrafish retinitis pigmentosa model.

Retinitis pigmentosa (RP) is a collection of heterogenous genetic retinal disorders resulting in cumulative retinal deterioration involving progressive loss of photoreceptors and eventually in total blindness. Oxidative stress plays a central role in this photoreceptor loss. Gypenosides (Gyp) are the main functional component isolated from the climbing vine Gynostemma pentaphyllum and have been shown to defend cells against the effects of oxidative stress and inflammation, providing protection in experimentally-induced optic neuritis. The zebrafish model has been used to investigate a range of human diseases. Previously we reported early retinal degeneration in a mutant zebrafish line carrying a point-nonsense mutation in the retinitis pigmentosa GTPase regulator interacting protein 1 (rpgrip1) gene that is mutated in RP patients. The current study investigated the potential protective effects of Gyp against photoreceptor degeneration in the Rpgrip1 deleted zebrafish. Rpgrip1 mutant zebrafish were treated with 5 µg/ml of Gyp in E3 medium from 6 h post fertilization (hpf) till 1 month post fertilization (mpf). Rpgrip1 mutant zebrafish treated with 5 µg/ml of Gyp showed a significant decrease by 68.41% (p = 0.0002) in photoreceptor cell death compared to that of untreated mutant zebrafish. Expression of antioxidant genes catalase, sod1, sod2, gpx1, gclm, nqo-1 and nrf-2 was significantly decreased in rpgrip1 mutant zebrafish eyes by 61.51%, 77.40%, 60.11%, 81.17%, 72.07%, 78.95% and 85.42% (all p < 0.0001), respectively, when compared to that of wildtype zebrafish; superoxide dismutase and catalase activities, and glutathione levels in rpgrip1 mutant zebrafish eyes were significantly decreased by 87.21%, 21.55% and 96.51% (all p < 0.0001), respectively. There were marked increases in the production of reactive oxygen species (ROS) and malondialdehyde (MDA) by 2738.73% and 510.69% (all p < 0.0001), respectively, in rpgrip1 mutant zebrafish eyes; expression of pro-inflammatory cytokines IL-1ß, IL-6 and TNF-α was also significantly increased by 150.11%, 267.79% and 190.72% (all p < 0.0001), respectively, in rpgrip1 mutant zebrafish eyes, compared to that of wildtype zebrafish. Treatment with Gyp significantly counteracted these effects. This study indicates that Gyp has a potential role in the treatment of RP.

Disease mechanisms and neuroprotection by tauroursodeoxycholic acid in Rpgr knockout mice.

Mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene are the predominant cause of retinitis pigmentosa. RPGR plays a critical role as a scaffold protein in the regulation of protein trafficking from the basal body to the axoneme, where the cargoes are transported to the outer segments (OSs) of photoreceptors. This trafficking process is controlled directly by intraflagellar transport complexes and regulated by the RPGR protein complex, although the precise mechanisms have yet to be defined. We used an Rpgr conditional knockout (cko) mouse model to investigate the disease mechanisms during retinal degeneration and to evaluate the protective effects of tauroursodeoxycholic acid (TUDCA). Rhodopsin, cone opsins and transducin were mislocalized in Rpgr cko photoreceptors, while localization of NPHP4 to connecting cilia was absent, suggesting that RPGR is required for ciliary protein trafficking. Microglia were activated in advance of retinal degeneration in Rpgr cko mouse retinas. TUDCA treatment suppressed microglial activation and inflammation and prevented photoreceptor degeneration in Rpgr cko mice. Our data demonstrated that TUDCA has therapeutic potential for RPGR-associated RP patients.

Retinal pigment epithelium cholesterol efflux mediated by the 18 kDa translocator protein, TSPO, a potential target for treating age-related macular degeneration.

Cholesterol accumulation beneath the retinal pigment epithelium (RPE) cells is supposed to contribute the pathogenesis of age-related macular degeneration (AMD). Cholesterol efflux genes (APOE and ABCA1) were identified as risk factors for AMD, although how cholesterol efflux influences accumulation of this lipid in sub-RPE deposits remains elusive. The 18 kDa translocator protein, TSPO, is a cholesterol-binding protein implicated in mitochondrial cholesterol transport. Here, we investigate the function of TSPO in cholesterol efflux from the RPE cells. We demonstrate in RPE cells that TSPO specific ligands promoted cholesterol efflux to acceptor (apo)lipoprotein and human serum, while loss of TSPO resulted in impaired cholesterol efflux. TSPO-/- RPE cells also had significantly increased production of reactive oxygen species (ROS) and upregulated expression of proinflammatory cytokines (IL-1ß and TNFα). Cholesterol (oxidized LDL) uptake and accumulation were markedly increased in TSPO-/- RPE cells. Finally, in aged RPE cells, TSPO expression was reduced and cholesterol efflux impaired. These findings provide a new pharmacological concept to treat early AMD patients by stimulating cellular cholesterol removal with TSPO specific ligands or by overexpression of TSPO in RPE cells.

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.

Deletion of TSPO Resulted in Change of Metabolomic Profile in Retinal Pigment Epithelial Cells.

Age-related macular degeneration is the main cause of vision loss in the aged population worldwide. Drusen, extracellular lesions formed underneath the retinal pigment epithelial (RPE) cells, are a clinical feature of AMD and associated with AMD progression. RPE cells support photoreceptor function by providing nutrition, phagocytosing outer segments and removing metabolic waste. Dysfunction and death of RPE cells are early features of AMD. The translocator protein, TSPO, plays an important role in RPE cholesterol efflux and loss of TSPO results in increased intracellular lipid accumulation and reactive oxygen species (ROS) production. This study aimed to investigate the impact of TSPO knockout on RPE cellular metabolism by identifying the metabolic differences between wildtype and knockout RPE cells, with or without treatment with oxidized low density lipoprotein (oxLDL). Using liquid chromatography mass spectrometry (LC/MS), we differentiated several metabolic pathways among wildtype and knockout cells. Lipids amongst other intracellular metabolites were the most influenced by loss of TSPO and/or oxLDL treatment. Glucose, amino acid and nucleotide metabolism was also affected. TSPO deletion led to up-regulation of fatty acids and glycerophospholipids, which in turn possibly affected the cell membrane fluidity and stability. Higher levels of glutathione disulphide (GSSG) were found in TSPO knockout RPE cells, suggesting TSPO regulates mitochondrial-mediated oxidative stress. These data provide biochemical insights into TSPO-associated function in RPE cells and may shed light on disease mechanisms in AMD.

Pathogenic mutations in retinitis pigmentosa 2 predominantly result in loss of RP2 protein stability in humans and zebrafish.

Mutations in retinitis pigmentosa 2 (RP2) account for 10-20% of X-linked retinitis pigmentosa (RP) cases. The encoded RP2 protein is implicated in ciliary trafficking of myristoylated and prenylated proteins in photoreceptor cells. To date >70 mutations in RP2 have been identified. How these mutations disrupt the function of RP2 is not fully understood. Here we report a novel in-frame 12-bp deletion (c.357_368del, p.Pro120_Gly123del) in zebrafish rp2 The mutant zebrafish shows reduced rod phototransduction proteins and progressive retinal degeneration. Interestingly, the protein level of mutant Rp2 is almost undetectable, whereas its mRNA level is near normal, indicating a possible post-translational effect of the mutation. Consistent with this hypothesis, the equivalent 12-bp deletion in human RP2 markedly impairs RP2 protein stability and reduces its protein level. Furthermore, we found that a majority of the RP2 pathogenic mutations (including missense, single-residue deletion, and C-terminal truncation mutations) severely destabilize the RP2 protein. The destabilized RP2 mutant proteins are degraded via the proteasome pathway, resulting in dramatically decreased protein levels. The remaining non-destabilizing mutations T87I, R118H/R118G/R118L/R118C, E138G, and R211H/R211L are suggested to impair the interaction between RP2 and its protein partners (such as ARL3) or with as yet unknown partners. By utilizing a combination of in silico, in vitro, and in vivo approaches, our work comprehensively indicates that loss of RP2 protein structural stability is the predominating pathogenic consequence for most RP2 mutations. Our study also reveals a role of the C-terminal domain of RP2 in maintaining the overall protein stability.

Carnosic acid attenuates acrylamide-induced retinal toxicity in zebrafish embryos.

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 µM) 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.

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.

TSPO Ligands Promote Cholesterol Efflux and Suppress Oxidative Stress and Inflammation in Choroidal Endothelial Cells.

Choroidal endothelial cells supply oxygen and nutrients to retinal pigment epithelial (RPE) cells and photoreceptors, recycle metabolites, and dispose of metabolic waste through the choroidal blood circulation. Death of the endothelial cells of the choroid may cause abnormal deposits including unesterified and esterified cholesterol beneath RPE cells and within Bruch's membrane that contribute to the progression of age-related macular degeneration (AMD), the most prevalent cause of blindness in older people. Translocator protein (TSPO) is a cholesterol-binding protein that is involved in mitochondrial cholesterol transport and other cellular functions. We have investigated the role of TSPO in choroidal endothelial cells. Immunocytochemistry showed that TSPO was localized to the mitochondria of choroidal endothelial cells. Choroidal endothelial cells exposed to TSPO ligands (Etifoxine or XBD-173) had significantly increased cholesterol efflux, higher expression of cholesterol homeostasis genes (LXRα, CYP27A1, CYP46A1, ABCA1 and ABCG1), and reduced biosynthesis of cholesterol and phospholipids from [14C]acetate, when compared to untreated controls. Treatment with TSPO ligands also resulted in reduced production of reactive oxygen species (ROS), increased antioxidant capacity, and reduced release of pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α and VEGF) induced by oxidized LDL. These data suggest TSPO ligands may offer promise for the treatment of AMD.

Knockout of RP2 decreases GRK1 and rod transducin subunits and leads to photoreceptor degeneration in zebrafish.

Retinitis pigmentosa (RP) affects about 1.8 million individuals worldwide. X-linked retinitis pigmentosa (XLRP) is one of the most severe forms of RP. Nearly 85% of XLRP cases are caused by mutations in the X-linked retinitis pigmentosa 2 (RP2) and RPGR. RP2 has been considered to be a GTPase activator protein for ARL3 and to play a role in the traffic of ciliary proteins. The mechanism of how RP2 mutations cause RP is still unclear. In this study, we generated an RP2 knockout zebrafish line using transcription activator-like effector nuclease technology. Progressive retinal degeneration could be observed in the mutant zebrafish. The degeneration of rods' outer segments (OSs) is predominant, followed by the degeneration of cones' OS. These phenotypes are similar to the characteristics of RP2 patients, and also partly consistent with the phenotypes of RP2 knockout mice and morpholino-mediated RP2 knockdown zebrafish. For the first time, we found RP2 deletion leads to decreased protein levels and abnormal retinal localizations of GRK1 and rod transducin subunits (GNAT1 and GNB1) in zebrafish. Furthermore, the distribution of the total farnesylated proteins in zebrafish retina is also affected by RP2 ablation. These molecular alterations observed in the RP2 knockout zebrafish might probably be responsible for the gradual loss of the photoreceptors' OSs. Our work identified the progression of retinal degeneration in RP2 knockout zebrafish, provided a foundation for revealing the pathogenesis of RP caused by RP2 mutations, and would help to develop potential therapeutics against RP in further studies.