Protective Effects of Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) Against Oxidative Stress in Zebrafish Hair Cells

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide, with known antiapoptotic functions. Our previous in vitro study has demonstrated the ameliorative role of PACAP-38 in chicken hair cells under oxidative stress conditions, but its effects on living hair cells is now yet known. Therefore, the aim of the present study was to investigate in vivo the protective role of PACAP-38 in hair cells found in zebrafish (Danio rerio) sense organs-neuromasts. To induce oxidative stress the 5-day postfertilization (dpf) zebrafish larvae were exposed to 1.5 mM H2O2 for 15 min or 1 h. This resulted in an increase in caspase-3 and p-38 MAPK level in the hair cells as well as in an impairment of the larvae basic behavior. To investigate the ameliorative role of PACAP-38, the larvae were incubated with a mixture of 1.5 mM H2O2 and 100 nM PACAP-38 following 1 h preincubation with 100 nM PACAP-38 only. PACAP-38 abilities to prevent hair cells from apoptosis were investigated. Whole-mount immunohistochemistry and confocal microscopy analyses revealed that PACAP-38 treatment decreased the cleaved caspase-3 level in the hair cells, but had no influence on p-38 MAPK. The analyses of basic locomotor activity supported the protective role of PACAP-38 by demonstrating the improvement of the fish behavior after PACAP-38 treatment. In summary, our in vivo findings demonstrate that PACAP-38 protects zebrafish hair cells from oxidative stress by attenuating oxidative stress-induced apoptosis.Peer reviewe

The Inhibitory Effect of Selected D2 Dopaminergic Receptor Agonists on VEGF-Dependent Neovascularization in Zebrafish Larvae: Potential New Therapy in Ophthalmic Diseases

Pathological angiogenesis is correlated with many ophthalmic diseases. The most common are exudative age-related macular degeneration and proliferative diabetic retinopathy. The current treatment for these diseases is based on regularly administered anti-VEGF antibodies injections. In the study, we investigated selected D2 dopaminergic receptor agonists, namely bromocriptine, cabergoline and pergolide, on hypoxia-induced neovascularization. We used the zebrafish laboratory model, specifically three-day post fertilization (dpf) Tg(fli-1: EGFP) zebrafish larvae. To induce abnormal angiogenesis of hyaloid-retinal vessels (HRVs) and intersegmental vessels (ISVs), the larvae were treated with cobalt chloride (II) (CoCl2) (a hypoxia-inducing agent) from 24 h post fertilization. The inhibitory role of D2 dopaminergic receptor agonists was investigated using confocal microscopy and qPCR. Additionally, the results were compared to those obtained in the group treated with CoCl2 followed by bevacizumab, the well-known antiangiogenic agent. Confocal microscopy analyses revealed severe deformation of vessels in the CoCl2 treated group, while co-incubation with bromocriptine, cabergoline, pergolide and bevacizumab, respectively, significantly inhibited abnormalities of angiogenesis. The qPCR analyses supported the protective role of the chosen dopaminergic agonists by demonstrating their influence on CoCl2-derived upregulation of vegfaa expression. The present results suggest that the D2 receptor agonists can be considered as a new direction in research for antiangiogenic therapy

P-Glycoprotein Inhibitor Tariquidar Plays an Important Regulatory Role in Pigmentation in Larval Zebrafish

Zebrafish has emerged as a powerful model in studies dealing with pigment development and pathobiology of pigment diseases. Due to its conserved pigment pattern with established genetic background, the zebrafish is used for screening of active compounds influencing melanophore, iridophore, and xanthophore development and differentiation. In our study, zebrafish embryos and larvae were used to investigate the influence of third-generation noncompetitive P-glycoprotein inhibitor, tariquidar (TQR), on pigmentation, including phenotype effects and changes in gene expression of chosen chromatophore differentiation markers. Five-day exposure to increasing TQR concentrations (1 µM, 10 µM, and 50 µM) resulted in a dose-dependent augmentation of the area covered with melanophores but a reduction in the area covered by iridophores. The observations were performed in three distinct regions—the eye, dorsal head, and tail. Moreover, TQR enhanced melanophore renewal after depigmentation caused by 0.2 mM 1-phenyl-2-thiourea (PTU) treatment. qPCR analysis performed in 56-h post-fertilization (hpf) embryos demonstrated differential expression patterns of genes related to pigment development and differentiation. The most substantial findings include those indicating that TQR had no significant influence on leukocyte tyrosine kinase, GTP cyclohydrolase 2, tyrosinase-related protein 1, and forkhead box D3, however, markedly upregulated tyrosinase, dopachrome tautomerase and melanocyte inducing transcription factor, and downregulated purine nucleoside phosphorylase 4a. The present study suggests that TQR is an agent with multidirectional properties toward pigment cell formation and distribution in the zebrafish larvae and therefore points to the involvement of P-glycoprotein in this process

A set of microphotographs and a graph documenting inhibition of neutrophil migration towards 10 µM CuSO₄ exposed hair cells resulting from co-treatment of 5 dpf Tg(MPX:GFP) zebrafish larvae (exhibiting green fluorescence in neutrophils) with 100 nM PACAP-38.

<p>(A) The control untreated larva presented normal distribution of neutrophils which were found in the ventral myotomes of the trunk and tail. (B) 10 µM CuSO₄ exposure evoked the migration of the immune cells towards the midline of the body and the formation of characteristic concentrations very close to, and around, the neuromasts (arrows). (C) 100 nM PACAP-38 co-treatment resulted in the inhibition of the neutrophil migration, which was reflected by a decreased number, or complete lack of, the green fluorescent cells in the area of natural neuromast localization (arrows). (D) 100 nM PACAP-38 itself did not visibly alter the natural distribution of neutrophils. The visualization was accomplished using a Zeiss LSM-700 confocal microscope. (E) The graph presenting the influence of 100 nM PACAP-38 on the number of the neutrophils concentrated around right posterior lateral line neuromasts (PLL) (L1, LII.1, L2, LII.2, L3, L4, L5 and L6) after 10 µM CuSO₄ exposure. The presented values refer to the average number of neutrophils in each group. 100 nM PACAP-38 treatment resulted in a significant, over two-fold decrease in the number of the neutrophils found singly in the area defined by notochord borders and those associated with neuromasts as compared to that determined in the 10 µM CuSO₄-exposed group (one-way ANOVA, Kruskal–Wallis test with Dunn’s post-test, GraphPad Prism 5, <i>p</i> < 0.001). N/group = 15.</p

A graph illustrating expression profiles of genes encoding three PACAP receptors (PAC1, VPAC1 and VPAC2) in samples from zebrafish 5 dpf larvae, kidney tissue and neutrophils.

<p>The material concerning larvae consisted of pooled individuals (n = 30), the kidney tissue refers to a single cell suspension derived from kidneys of five adult zebrafish and the neutrophil population consisting of 500,000 GFP+ sorted cells by FACS. Each collection was covered by samples analyzed in triplicate. Data in the figure represent the average of the representative experiment. Gene expression values were normalized to housekeeping gene β-actin. The existence of all types of PACAP receptor transcripts was reported in each collection, however at a different levels. In neutrophils, <i>adcyap1r1a</i> appeared as a predominant form for PAC1 and, for the VPAC family, the most prevalent was <i>vipr1b</i>. The lowest expression level was exhibited by <i>adcyap1r1b</i> and <i>vipr1a</i> genes. Interestingly, <i>adcyap1r1a</i> and <i>vipr1b</i> were expressed at similar levels in each studied sample, whereas for the remaining PACAP receptor genes, the highest expression levels were found in whole larvae, lower in the kidney tissue and the lowest was in neutrophils.</p

Pituitary adenylate cyclase–activating polypeptide (PACAP-38) plays an inhibitory role against inflammation induced by chemical damage to zebrafish hair cells

<div><p>Pituitary adenylate cyclase–activating polypeptide (PACAP-38) is a common neuropeptide exerting a wide spectrum of functions in many fields, including immunology. In the present study, 5-day post-fertilization (dpf) zebrafish larvae of three diverse genetic lines [transgenic lines Tg(MPX:GFP) with GFP-labelled neutrophils and Tg(pou4f3:GAP-GFP) with GFP-labelled hair cells and the wild-type Tuebingen] were used to investigate an inhibitory role of PACAP-38 in inflammation associated with damaged hair cells of the lateral line. Individuals of each genetic line were assigned to four groups: (1) control, and those consisting of larvae exposed to (2) 10 µM CuSO₄, (3) 10 µM CuSO₄+100 nM PACAP-38 and (4) 100 nM PACAP-38, respectively. Forty-minute exposure to CuSO₄ solution was applied to evoke necrosis of hair cells and consequent inflammation. The inhibitory role of PACAP-38 was investigated <i>in vivo</i> under a confocal microscope by counting neutrophils migrating towards damaged hair cells in Tg(MPX:GFP) larvae. In CuSO₄-treated individuals, the number of neutrophils associated with hair cells was dramatically increased, while PACAP-38 co-treatment resulted in its over 2-fold decrease. However, co-treatment with PACAP-38 did not prevent hair cells from extensive necrosis, which was found in Tg(pou4f3:GAP-GFP) individuals. Real-Time PCR analysis performed in wild-type larvae demonstrated differential expression pattern of stress and inflammation inducible markers. The most significant findings showed that CuSO₄ exposure up-regulated the expression of <i>IL-8</i>, <i>IL-1β</i>, <i>IL-6</i> and <i>ATF3</i>, while after PACAP-38 co-treatment expression levels of these genes were significantly decreased. The presence of transcripts for all PACAP receptors in neutrophils was also revealed. <i>Adcyap1r1a</i> and <i>vipr1b</i> appeared to be predominant forms. The present results suggest that PACAP-38 should be considered as a factor playing an important regulatory role in inflammatory response associated with pathological processes affecting zebrafish hair cells and it cannot be excluded that this interesting property has more universal significance.</p></div

Expression profiles of pro-inflammatory and stress-inducible genes.

<p>The graphs present the mRNA expression of (A) interleukin 1β (<i>IL-1β</i>), (B) interleukin 6 (<i>IL-6</i>), (C) interleukin 8 (<i>IL-8</i>), (D) interleukin 10 (<i>IL-10</i>), (E) macrophage receptor MARCO (<i>MARCO</i>), (F) activating transcription factor 3 (<i>ATF3</i>), (G) tumor necrosis factor (<i>TNFα</i>), (H) C-X-C motif chemokine receptor 1 (<i>CXCR1</i>) and (I) C-X-C motif chemokine receptor 2 (<i>CXCR2</i>) from pooled (n = 30) 5dpf wild-type zebrafish larvae in four experimental groups: 1) control, 2) exposed to 10µM CuSO₄ for 40 min, 3) exposed to a mixture of 100 nM PACAP-38 + 10µM CuSO₄ for 40 min preceded by one hour pre-incubation with 100 nM PACAP-38 only, and 4) exposed to 100nM PACAP-38 only. Each group was covered by samples analyzed in triplicate in three separate experiments. Data in the figure represent the average of the three individual experiments. Gene expression values were normalized to housekeeping gene <i>β-actin</i>. To maintain image clarity, only differences between CuSO₄ exposed and PACAP-38 co-treated groups are marked on the graphs. Co-treatment with PACAP-38 significantly reduced up-regulated by copper treatment <i>IL-1β</i> and <i>IL-6</i>, <i>IL-8</i> and <i>ATF3</i> gene expressions (A, B, C and F) (one-way ANOVA, Kruskal–Wallis test with Dunn’s posttest, GraphPad Prism 5, <i>p</i> < 0.05), while it had no significant influence on <i>IL-10</i>, <i>MARCO</i> or <i>TNFα</i> genes (D, E and G) (one-way ANOVA, Kruskal–Wallis test with Dunn’s posttest, GraphPad Prism 5, <i>p</i> > 0.05). With regard to <i>CXCR1</i> and <i>CXCR2</i>, 10 µM copper chemical injury and 100 nM PACAP-38 treatment did not significantly change the expression level of the receptors. (one-way ANOVA, Kruskal–Wallis test with Dunn’s post-test, GraphPad Prism 5, <i>p</i> > 0.05).</p

Primers used in the study.

<p>Primers used in the study.</p

A Zebrafish/Drosophila Dual System Model for Investigating Human Microcephaly

Microcephaly presents in neurodevelopmental disorders with multiple aetiologies, including bi-allelic mutation in TUBGCP2, a component of the biologically fundamental and conserved microtubule-nucleation complex, &gamma;-TuRC. Elucidating underlying principles driving microcephaly requires clear phenotype recapitulation and assay reproducibility, areas where go-to experimental models fall short. We present an alternative simple vertebrate/invertebrate dual system to investigate fundamental TUBGCP2-related processes driving human microcephaly and associated developmental traits. We show that antisense morpholino knockdown (KD) of the Danio rerio homolog, tubgcp2, recapitulates human TUBGCP2-associated microcephaly. Co-injection of wild type mRNA pre-empts microcephaly in 55% of KD zebrafish larvae, confirming causality. Body shortening observed in morphants is also rescued. Mitotic marker (pH3) staining further reveals aberrantly accumulated dividing brain cells in microcephalic tubgcp2 KD morphants, indicating that tubgcp2 depletion disrupts normal mitosis and/or proliferation in zebrafish neural progenitor brain cells. Drosophila melanogaster double knockouts (KO) for TUBGCP2 homologs Grip84/cg7716 also develop microcephalic brains with general microsomia. Exacerbated Grip84/cg7716-linked developmental aberration versus single mutations strongly suggests interactive or coinciding gene functions. We infer that tubgcp2 and Grip84/cg7716 affect brain size similarly to TUBGCP2 and recapitulate both microcephaly and microcephaly-associated developmental impact, validating the zebrafish/fly research model for human microcephaly. Given the conserved cross-phyla homolog function, the data also strongly support mitotic and/or proliferative disruption linked to aberrant microtubule nucleation in progenitor brain cells as key mechanistic defects for human microcephaly