Estrogenic regulation of claudin 5 and tight junction protein 1 gene expression in zebrafish: A role on blood-brain barrier?

The blood-brain barrier (BBB) is a physical interface between the blood and the brain parenchyma, playing key roles in brain homeostasis. In mammals, the BBB is established thanks to tight junctions between cerebral endothelial cells, involving claudin, occludin, and zonula occludens proteins. Estrogens have been documented to modulate BBB permeability. Interestingly, in the brain of zebrafish, the estrogen-synthesizing activity is strong due to the high expression of Aromatase B protein, encoded by the cyp19a1b gene, in radial glial cells (neural stem cells). Given the roles of estrogens in BBB function, we investigated their impact on the expression of genes involved in BBB tight junctions. We treated zebrafish embryos and adult males with 17ß-estradiol and observed an increased cerebral expression of tight junction and claudin 5 genes in adult males only. In females, treatment with the nuclear estrogen receptor antagonist (ICI182,780 ) had no impact. Interestingly, telencephalic injuries performed in males decreased tight junction gene expression that was partially reversed with 17ß-estradiol. This was further confirmed by extravasation experiments of Evans blue showing that estrogenic treatment limits BBB leakage. We also highlighted the intimate links between endothelial cells and neural stem cells, suggesting that cholesterol and peripheral steroids could be taken up by endothelial cells and used as precursors for estrogen synthesis by neural stem cells. Together, our results show that zebrafish provides an alternative model to further investigate the role of steroids on the expression of genes involved in BBB integrity, both in constitutive and regenerative physiological conditions. The link we described between capillaries endothelial cells and steroidogenic neural cells encourages the use of this model in understanding the mechanisms by which peripheral steroids get into neural tissue and modulate neurogenic activity.

Insulin signaling promotes neurogenesis in the brain of adult zebrafish.

Insulin is a peptide hormone that plays a central role in the regulation of circulating blood glucose in vertebrates, including zebrafish. Increasing evidence has demonstrated the important role of insulin in many brain functions. In zebrafish, two insulin receptor genes (insra and insrb) have been identified. However, their biodistribution in the adult brain as well as their cell-specific expression pattern has not been well described. Using gene expression analysis, in situ hybridization and transgenic fish, we confirmed the expression of insra, insrb, and irs1 (insulin receptor substrate 1, the downstream effector of insulin receptor) in the brain of adult zebrafish and characterized their specific expression in neurons and neural stem cells (radial glia). After demonstrating that intracerebroventricular (ICV) injection resulted in the diffusion of the injected solution within the ventricular system, we analyzed the effect of insulin ICV injection on neurogenesis. We showed that insulin promotes ventricular cell proliferation 24 h postinjection. This neurogenic effect appeared to be independent of neuroinflammatory processes. Also, after a mechanical telencephalic stab-wound injury, we highlighted the overexpression of irs1 gene 5 days postlesion notably in the ventricular zone where radial glial cells (RGCs) are localized, suggesting key roles of insulin signaling in regenerative processes. Finally, our results reinforced the expression of insulin-related proteins in the brain of adult zebrafish, highlighting the potential role of insulin signaling on neurogenesis.

Distribution of microglia/immune cells in the brain of adult zebrafish in homeostatic and regenerative conditions: Focus on oxidative stress during brain repair.

Microglia are macrophage-like cells exerting determinant roles in neuroinflammatory and oxidative stress processes during brain regeneration. We used zebrafish as a model of brain plasticity and repair. First, by performing L-plastin (Lcp1) immunohistochemistry and using transgenic Tg(mpeg1.1:GFP) or Tg(mpeg1.1:mCherry) fish, we analyzed the distribution of microglia/immune cells in the whole brain. Specific regional differences were evidenced in terms of microglia/immune cell density and morphology (elongated, branched, highly branched, and amoeboid). Taking advantage of Tg(fli:GFP) and Tg(GFAP::GFP) enabling the detection of endothelial cells and neural stem cells (NSCs), we highlighted the association of elongated microglia/immune cells with blood vessels and rounded/amoeboid microglia with NSCs. Second, after telencephalic injury, we showed that L-plastin cells were still abundantly present at 5 days post-lesion (dpl) and were associated with regenerative neurogenesis. Finally, RNA-sequencing analysis from injured telencephalon (5 dpl) confirmed the upregulation of microglia/immune cell markers and highlighted a significant increase of genes involved in oxidative stress (nox2, nrf2a, and gsr). The analysis of antioxidant activities at 5 dpl also revealed an upregulation of superoxide dismutase and persistent H2 O2 generation in the injured telencephalon. Also, microglia/immune cells were shown to be a source of oxidative stress at 5 dpl. Overall, our data provide a better characterization of microglia/immune cell distribution in the healthy zebrafish brain, highlighting some evolutionarily conserved features with mammals. They also emphasize that 5 days after injury, microglia/immune cells are still activated and are associated to a persistent redox imbalance. Together, these data raise the question of the role of oxidative stress in regenerative neurogenesis in zebrafish.

Hypericum lanceolatum Lam. Medicinal Plant: Potential Toxicity and Therapeutic Effects Based on a Zebrafish Model.

Hypericum lanceolatum Lam. (H. lanceolatum) is a traditional medicinal plant from Reunion Island used for its pleiotropic effects mainly related to its antioxidant activity. The present work aimed to 1) determine the potential toxicity of the plant aqueous extract in vivo and 2) investigate its putative biological properties using several zebrafish models of oxidative stress, regeneration, estrogenicity, neurogenesis and metabolic disorders. First, we characterized the polyphenolic composition by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and identified chlorogenic acid isomers, quercetin and kaempferol derivatives as the major compounds. We then evaluated for the first time the toxicity of an aqueous extract of H. lanceolatum and determined a maximum non-toxic concentration (MNTC) in zebrafish eleutheroembryos from 0 to 96 hpf following OECD (Organization for Economic Cooperation and Development) guidelines. This MNTC test was also determined on hatched eleutheroembryos after 2 days of treatment (from 3 to 5 dpf). In our study, the anti-estrogenic effects of H. lanceolatum are supported by the data from the EASZY assay. In a tail amputation model, we showed that H. lanceolatum at its MNTC displays antioxidant properties, favors immune cell recruitment and tissue regeneration. Our results also highlighted its beneficial effects in metabolic disorders. Indeed, H. lanceolatum efficiently reduces lipid accumulation and body mass index in overfed larva- and adult-models, respectively. In addition, we show that H. lanceolatum did not improve fasting blood glucose levels in a hyperglycemic zebrafish model but surprisingly inhibited neurogenesis impairment observed in diabetic conditions. In conclusion, our study highlights the antioxidant, pro-regenerative, anti-lipid accumulation and pro-neurogenic effects of H. lanceolatum in vivo and supports the use of this traditional medicinal plant as a potential alternative in the prevention and/or treatment of metabolic disorders.