The generation and characterization of a transgenic zebrafish line with lens-specific Cre expression.

Purpose: Danio rerio zebrafish constitute a popular model for studying lens development and congenital cataracts. However, the specific deletion of a gene with a Cre/LoxP system in the zebrafish lens is unavailable because of the lack of a lens-Cre-transgenic zebrafish. This study aimed to generate a transgenic zebrafish line in which Cre recombinase was specifically expressed in the lens. Methods: The pTol2 cryaa:Cre-polyA-cryaa:EGFP (enhanced green fluorescent protein) plasmid was constructed and co-injected with Tol2-transposase into one-to-two-cell-stage wild-type (WT) zebrafish embryos. Whole-mount in situ hybridization (ISH), tissue section, hematoxylin and eosin staining, a Western blot, a split-lamp observation, and a grid transmission assay were used to analyze the Cre expression, lens structure, and lens transparency of the transgenic zebrafish. Results: In this study, we generated a transgenic zebrafish line, zTg(cryaa:Cre-cryaa:EGFP), in which Cre recombinase and EGFP were driven by the lens-specific cryaa promoter. zTg(cryaa:Cre-cryaa:EGFP) began to express Cre and EGFP specifically in the lens at the 22 hpf stage, and this ectopic Cre could efficiently and specifically delete the red fluorescent protein (RFP) signal from the lens when zTg(cryaa:Cre-cryaa:EGFP) embryos were injected with the loxP-flanked RFP plasmid. The overexpression of Cre and EGFP did not impair zebrafish development or lens transparency. Accordingly, this zTg(cryaa:Cre-cryaa:EGFP) zebrafish line is a useful tool for gene editing, specifically with zebrafish lenses. Conclusions: We established a zTg(cryaa:Cre-cryaa:EGFP) zebrafish line that can specifically express an active Cre recombinase in lens tissues. This transgenic zebrafish line can be used as a tool to specifically manipulate a gene in zebrafish lenses.

The downregulation of HSP90-controlled CRALBP expression is associated with age-related vision attenuation.

The dysfunction of CRALBP, a key regulator of the visual cycle, is associated with retinitis punctata albescens characterized by night vision loss and retinal degeneration. In this paper, we find that the expression of CRALBP is regulated by heat shock protein 90 (HSP90). Inhibition of HSP90α or HSP90ß expression by using the CRISPR-Cas9 technology downregulates CRALBP's mRNA and protein expression in ARPE-19 cells by triggering the degradation of transcription factor SP1 in the ubiquitin-proteasome pathway. SP1 can bind to CRALBP's promoter, and inhibition of SP1 by its inhibitor plicamycin or siRNA downregulates CRALBP's mRNA expression. In the zebrafish, inhibition of HSP90 by the intraperitoneal injection of IPI504 reduces the thickness of the retinal outer nuclear layer and Rlbp1b mRNA expression. Interestingly, the expression of HSP90, SP1, and CRALBP is correlatedly downregulated in the senescent ARPE-19 and Pig primary RPE cells in vitro and in the aged zebrafish and mouse retinal tissues in vivo. The aged mice exhibit the low night adaption activity. Taken together, these data indicate that the HSP90-SP1 is a novel regulatory axis of CRALBP transcriptional expression in RPE cells. The age-mediated downregulation of the HSP90-SP1-CRALBP axis is a potential etiology for the night vision reduction in senior people.

SARS-COV-2 spike protein promotes RPE cell senescence via the ROS/P53/P21 pathway.

SARS-Cov-2 infection, which has caused the COVID-19 global pandemic, triggers cellular senescence. In this study, we investigate the role of the SARS-COV-2 spike protein (S-protein) in regulating the senescence of RPE cells. The results showed that administration or overexpression of S-protein in ARPE-19 decreased cell proliferation with cell cycle arrest at the G1 phase. S-protein increased SA-ß-Gal positive ARPE-19 cells with high expression of P53 and P21, senescence-associated inflammatory factors (e.g., IL-1ß, IL-6, IL-8, ICAM, and VEGF), and ROS. Elimination of ROS by N-acetyl cysteine (NAC) or knocking down p21 by siRNA diminished S-protein-induced ARPE cell senescence. Both administrated and overexpressed S-protein colocalize with the ER and upregulate ER-stress-associated BIP, CHOP, ATF3, and ATF6 expression. S-protein induced P65 protein nuclear translocation. Inhibition of NF-κB by bay-11-7082 reduced S-protein-mediated expression of senescence-associated factors. Moreover, the intravitreal injection of S-protein upregulates senescence-associated inflammatory factors in the zebrafish retina. In conclusions, the S-protein of SARS-Cov-2 induces cellular senescence of ARPE-19 cells in vitro and the expression of senescence-associated cytokines in zebrafish retina in vivo likely by activating ER stress, ROS, and NF-κb. These results may uncover a potential association between SARS-cov-2 infection and development of AMD.

Ghrelin modulates dopaminergic neuron formation and attention deficit hyperactivity disorder-like behaviors: From animals to human models.

Attention deficit hyperactivity disorder (ADHD) is one of the most prevalent psychiatric disorders in children. The orexigenic hormone ghrelin is important in neuroprotection and neurodevelopment, which may play an important role in psychopathogenesis of ADHD. This study aimed to systematically investigate the genomic and pharmacological manipulations of ghrelin functioning in ADHD-like symptoms in zebrafish models and validated the effects of ghrelin polymorphisms in human subjects with ADHD. We firstly generated ghrelinΔ/Δ zebrafish mutant, which displayed hyperactive, attention deficit-like and impulsive-like behaviors, as well as endophenotypes, mimicking human ADHD. GhrelinΔ/Δ zebrafish exhibited downregulated expression levels of wnt1, wnt3a, wnt5a that are critical for dopaminergic neuron development to possibly regulate their number and spatial organization. Pharmacological blockade of wnt signaling with XAV939 induced a reduced moving activity and less dopaminergic neurons; whereas, wnt agonist SB415286 rescued hyperactivity and dopaminergic neuron loss in ghrelinΔ/Δ zebrafish. In addition, we further identified and validated a SNP, rs696217, on orexigenic hormone preproghrelin/ghrelin (T408T, Met72Met) to be associated with a higher risk of ADHD in a case-controlled association study with 248 subjects with ADHD and 208 subjects of healthy controls. Together, our results reveal a novel endogenous role for orexigenic hormone ghrelin in ADHD, which provides insights into genetic regulation and drug screens for the identification of novel treatments of ADHD.

Steroidogenic acute regulatory protein and luteinizing hormone are required for normal ovarian steroidogenesis and oocyte maturation in zebrafish†.

In recent studies, luteinizing hormone (LH) was reported to play important roles in oocyte maturation. However, the mechanism by which LH signaling, especially regarding the steroidogenesis process, affects oocyte maturation has not been clarified. In this study, zebrafish models with a functional deficiency in luteinizing hormone beta (Lhb) or steroidogenic acute regulatory protein (Star), an enzyme that promotes the transport of cholesterol into the inner mitochondrial membrane for maturation-induced hormone (MIH) production, were generated using transcription activator-like effector nucleases (TALENs). Similar phenotypes of the maturation-arrested oocytes in both female mutants have been observed. The levels of MIH in the oocytes of the female mutants were clearly decreased in both the lhb and star knockout zebrafish. The expression of star was dramatically down-regulated in the lhb mutant follicles and was clearly promoted by forskolin and hCG in vitro. Furthermore, treatment with the MIH precursors, pregnenolone or progesterone, as well as with MIH itself rescued the maturation-arrested oocyte phenotypes in both lhb and star mutants. The plasma levels of other steroids, including testosterone, estradiol, and cortisol, were not affected in the lhb mutants, while the levels of gonad hormones testosterone and estradiol were significantly increased in the star mutants. The cortisol levels were decreased in the star mutants. Collectively, our results confirm that LH plays important roles in the initiation of MIH synthesis from cholesterol and maintains oocyte maturation in zebrafish, as well as provide evidence that Star might act downstream of LH signaling in steroidogenesis.

Different physiological roles of insulin receptors in mediating nutrient metabolism in zebrafish.

Insulin, the most potent anabolic hormone, is critical for somatic growth and metabolism in vertebrates. Type 2 diabetes, which is the primary cause of hyperglycemia, results from an inability of insulin to signal glycolysis and gluconeogenesis. Our previous study showed that double knockout of insulin receptor a ( insra) and b ( insrb) caused ß-cell hyperplasia and lethality from 5 to 16 days postfertilization (dpf) (Yang BY, Zhai G, Gong YL, Su JZ, Han D, Yin Z, Xie SQ. Sci Bull (Beijing) 62: 486-492, 2017). In this study, we characterized the physiological roles of Insra and Insrb, in somatic growth and fueling metabolism, respectively. A high-carbohydrate diet was provided for insulin receptor knockout zebrafish from 60 to 120 dpf to investigate phenotype inducement and amplification. We observed hyperglycemia in both insra-/- fish and insrb-/- fish. Impaired growth hormone signaling, increased visceral adiposity, and fatty liver were detected in insrb-/- fish, which are phenotypes similar to the lipodystrophy observed in mammals. More importantly, significantly diminished protein levels of P-PPARα, P-STAT5, and IGF-1 were also observed in insrb-/- fish. In insra-/- fish, we observed increased protein content and decreased lipid content of the whole body. Taken together, although Insra and Insrb show overlapping roles in mediating glucose metabolism through the insulin-signaling pathway, Insrb is more prone to promoting lipid catabolism and protein synthesis through activation of the growth hormone-signaling pathway, whereas Insra primarily acts to promote lipid synthesis via glucose utilization.

Enhanced insulin activity achieved in VDRa/b ablation zebrafish.

Introduction: 1α,25-dihydroxyvitamin D3 (1α,25[OH]2VD3) is a hormone known for its key roles in calcium absorption and nutrient metabolism. In teleost fishes, 1α,25(OH)2VD3 insufficiency causes impaired glucose metabolism and lipid oxidation. However, the cascade and mechanisms of 1α,25(OH)2VD3 and the vitamin d receptor (VDR) signaling are unclear. Results: In this study, two genes (vdra and vdrb) encoding paralogs of VDRs were genetically knocked out in zebrafish. Growth retardation and accumulated visceral adipose tissue have been observed in vdra -/-;vdrb -/- deficient line. In the liver elevated accumulation of triglycerides and suppressed lipid oxidation were detected. Morover significantly elevated 1α,25(OH)2VD3 levels were detected in vdra-/-;vdrb-/- zebrafish due to cyp24a1 transcription repression. Furthermore VDRs ablation Enhanced insulin signaling including elevated insulin/insra trancriptional levels, glycolysis, lipogenesis and promoted AKT/mTOR activity. Discussion: In conclusion, our present studies provides a zebrafish model with an elevated 1α,25(OH)2VD3 levels in vivo. The 1α,25(OH)2VD3/VDRs signaling promote lipid oxidation activity. However 1α,25(OH)2VD3 activity of regulation of glucose homeostasis through Insulin/Insr was independent of nuclear VDRs in teleosts.

Deficiency of heat shock factor 4 promotes lens epithelial cell senescence through upregulating p21cip1 expression.

Genetic mutations in heat shock factor 4 (Hsf4) is associated with both congenital and age-related cataracts. Hsf4 regulates lens development through its ability to both activate and inhibit transcription. Previous studies suggested Hsf4 is involved in modulating cellular senescence depending on p21cip1 and p27 kip1 expression in MEF cells. Here, we found that Hsf4 acts as a suppressor of p21cip1 expression and plays an anti-senescence role during lens development. Knocking out Hsf4 facilitated UVB-induced cellular senescence in mouse lens epithelial cells (mLECs). p21cip1 was upregulated at both the mRNA and protein levels in HSF4-/- mLECs under control and UVB-treated conditions, and knockdown of p21cip1 by siRNA alleviated UVB-induced cellular senescence. HSF4 directly bound to the p21cip1 promoter and increased H3K27m3 levels at the p21cip1 proximal promoter region by recruiting the methyltransferase EZH2. In animal models, p21cip1 was gradually upregulated in wild-type mouse lenses with increasing age, while Hsf4 levels decreased. We generated a Hsf4 mutant mice line (Hsf4del-42) which displayed obvious congenital cataract phenotype. The expression of p21cip1 and senescence-associated cytokines were induced in the cataractous lenses of Hsf4del-42 mice. H3K27m3 and EZH2 levels decreased in p21cip1 promoters in the lenses of Hsf4del-42 mice. The SA-ß-Gal activities were positive in lens epithelia of aged Hsf4null zebrafish compared to wild-type lenses. p21cip1 and senescence-associated cytokines levels were also upregulated in lenses of Hsf4null zebrafish. Accordingly, we propose that HSF4 plays a protective role in lens epithelial cells against cellular senescence during lens development and aging, partly by fine-tuning p21cip1 expression.

HSP90 acts as a senomorphic target in senescent retinal pigmental epithelial cells.

The senescence of retinal pigment epithelial (RPE) cells is associated with age-related macular degeneration (AMD), a leading cause of blindness in the world. HSP90 is a predominant chaperone that regulates cellular homeostasis under divergent physio-pathological conditions including senescence. However, the role of HSP90 in senescent RPE cells still remains unclear. Here, we reported that HSP90 acts as a senomorphic target of senescent RPE cells in vitro. Using H2O2-induced senescent ARPE-19 cells and replicative senescent primary RPE cells from rhesus monkey, we found that HSP90 upregulates the expression of IKKα, and HIF1α in senescent ARPE-19 cells and subsequently controls the induction of distinct senescence-associated inflammatory factors. Senescent ARPE-19 cells are more resistant to the cytotoxic HSP90 inhibitor IPI504 (IC50 = 36.78 µM) when compared to normal ARPE-19 cells (IC50 = 6.16 µM). Administration of IPI504 at 0.5-5 µM can significantly inhibit the induction of IL-1ß, IL-6, IL-8, MCP-1 and VEGFA in senescent ARPE-19 and the senescence-mediated migration of retinal capillary endothelial cells in vitro. In addition, we found that inhibition of HSP90 by IPI504 reduces SA-ß-Gal's protein expression and enzyme activity in a dose-dependent manner. HSP90 interacts with and regulates SA-ß-Gal protein stabilization in senescent ARPE-19 cells. Taken together, these results suggest that HSP90 regulates the SASP and SA-ß-Gal activity in senescent RPE cells through associating with distinctive mechanism including NF-κB, HIF1α and lysosomal SA-ß-Gal. HSP90 inhibitors (e.g. IPI504) could be a promising senomorphic drug candidate for AMD intervention.

Fatty Acid Oxidation in Zebrafish Adipose Tissue Is Promoted by 1α,25(OH)2D3.

1α,25(OH)2D3 (vitamin D3) is crucial for mineral homeostasis in mammals, but the precise effects of 1α,25(OH)2D3 in adipose tissue remain to be clarified in vivo. The initial 25-hydroxylation is catalyzed by liver microsomal cytochrome P450 2R1 (CYP2R1), which is conserved in vertebrates. To probe the physiological function(s) of 1α,25(OH)2D3 in teleosts, we generated two independent cyp2r1-deficient zebrafish lines. These mutants exhibit retarded growth and increased obesity, especially in the visceral adipose tissue (VAT). These defects could be rescued with 25(OH)D3 treatments. ChIP-PCR analyses demonstrated that pgc1a is the target of the vitamin D receptor in the liver and VAT of zebrafish. Significantly decreased protein levels of Pgc1a, impaired mitochondrial biogenesis, and free fatty acid oxidation are also observed in the cyp2r1 mutant VAT. Our results demonstrate that regulation of 1α,25(OH)2D3 during lipid metabolism occurs through the regulation of Pgc1a for mitochondrial biogenesis and oxidative metabolism within zebrafish VAT.

Hyperplasia and cellularity changes in IGF-1-overexpressing skeletal muscle of crucian carp.

The zebrafish skeletal muscle-specific promoter mylz2 was used to cause crucian carp overexpression of the zebrafish IGF-1 cDNA. In stable transgenic germline F1 progenies, a 5-fold increase in the level of IGF-1 in skeletal muscle was observed. Evident skeletal muscle hyperplasia was observed in the transgenic fish through histologic analysis. By analyzing the RNA sequencing transcriptome of the skeletal muscle of IGF-1 transgenic fish and nontransgenic control fish at 15 months of age, 10 966 transcripts with significant expression levels were identified with definite gene descriptions based on the corresponding zebrafish genome information. Based on the results of our RNA sequencing transcriptome profiling analysis and the results of the real-time quantitative PCR analysis performed to confirm the skeletal muscle transcriptomics analysis, several pathways, including IGF-1 signaling, aerobic metabolism, and protein degradation, were found to be activated in the IGF-1-overexpressing transgenic fish. Intriguingly, our transcriptional expression and protein assays indicated that the overexpression of IGF-1 stimulated a significant shift in the myofiber type toward a more oxidative slow muscle type. Although the body weight was surprisingly decreased by IGF-1 transgenic expression, significantly higher oxygen consumption rates were measured in IGF-1-overexpressing transgenic fish compared with their nontransgenic control fish. These results indicate that the sustained overexpression of IGF-1 in crucian carp skeletal muscle promotes myofiber hyperplasia and cellularity changes, which elicit alterations in the body energy metabolism and skeletal muscle growth.