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1.
Ecotoxicol Environ Saf ; 208: 111716, 2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33396047

RESUMO

Although withdrawn from the market in the 1980s, polychlorinated biphenyls (PCBs) are still found ubiquitously in the aquatic environment and pose a serious risk to biota due to their teratogenic potential. In fish, early life-stages are often considered most sensitive with regard to their exposure to PCBs and other dioxin-like compounds. However, little is known about the molecular drivers of the frequently observed teratogenic effects. Therefore, the aims of our study were to: (1) characterize the baseline transcriptome profiles at different embryonic life-stages in zebrafish (Danio rerio); and (2) to identify the molecular response to PCB exposure and life-stage specific-effects of the chemical on associated processes. For both objectives, embryos were sampled at 12, 48, and 96 h post-fertilization (hpf) and subjected to Illumina sequence-by-synthesis and RNAseq analysis. Results revealed that with increasing age more genes and related pathways were upregulated both in terms of number and magnitude. Yet, other transcripts followed an opposite pattern with greater transcript abundance at the earlier time points. Additionally, embryos were exposed to PCB126, a potent agonist of the aryl hydrocarbon receptor (AHR). ClueGO network analysis revealed significant enrichment of genes associated with basic cell metabolism, communication, and homeostasis as well as eye development, muscle formation, and skeletal formation. We selected eight genes involved in the affected pathways for an in-depth characterization of their regulation throughout normal embryogenesis and after exposure to PCB126 by quantification of transcript abundances every 12 h until 118 hpf. Among these, fgf7 and c9 stood out because of their strong upregulation by PCB126 exposure at 48 and 96 hpf, respectively. Cyp2aa12 was upregulated from 84 hpf on. Fabp10ab, myhz1.1, col8a1a, sulf1, and opn1sw1 displayed specific regulation depending on the developmental stage. Overall, we demonstrate that (1) the developmental transcriptome of zebrafish is highly dynamic, and (2) dysregulation of gene expression by exposure to PCB126 was significant and in several cases not directly connected to AHR-signaling. Hence, this study improves the understanding of linkages between molecular events and apical outcomes that are of regulatory relevance.


Assuntos
Desenvolvimento Embrionário/efeitos dos fármacos , Bifenilos Policlorados/toxicidade , Teratogênios/toxicidade , Poluentes Químicos da Água/toxicidade , Peixe-Zebra/embriologia , Animais , Embrião não Mamífero/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Transcriptoma , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores
2.
Int J Mol Sci ; 21(24)2020 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-33352756

RESUMO

Transcriptional changes normally occur during development but also underlie differences between healthy and pathological conditions. Transcription factors or chromatin modifiers are involved in orchestrating gene activity, such as the cohesin genes and their regulator NIPBL. In our previous studies, using a zebrafish model for nipblb knockdown, we described the effect of nipblb loss-of-function in specific contexts, such as central nervous system development and hematopoiesis. However, the genome-wide transcriptional impact of nipblb loss-of-function in zebrafish embryos at diverse developmental stages remains under investigation. By RNA-seq analyses in zebrafish embryos at 24 h post-fertilization, we examined genome-wide effects of nipblb knockdown on transcriptional programs. Differential gene expression analysis revealed that nipblb loss-of-function has an impact on gene expression at 24 h post fertilization, mainly resulting in gene inactivation. A similar transcriptional effect has also been reported in other organisms, supporting the use of zebrafish as a model to understand the role of Nipbl in gene regulation during early vertebrate development. Moreover, we unraveled a connection between nipblb-dependent differential expression and gene expression patterns of hematological cell populations and AML subtypes, enforcing our previous evidence on the involvement of NIPBL-related transcriptional dysregulation in hematological malignancies.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/genética , Animais , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Embrião não Mamífero/citologia , Perfilação da Expressão Gênica , Genoma , Peixe-Zebra/crescimento & desenvolvimento , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética
3.
J Neurosci ; 40(18): 3631-3645, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32245827

RESUMO

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that play critical roles in neuronal development and nervous system function. Here, we developed a model to study NMDARs in early development in zebrafish, by generating CRISPR-mediated lesions in the NMDAR genes, grin1a and grin1b, which encode the obligatory GluN1 subunits. While receptors containing grin1a or grin1b show high Ca2+ permeability, like their mammalian counterpart, grin1a is expressed earlier and more broadly in development than grin1b Both grin1a -/- and grin1b -/- zebrafish are viable. Unlike in rodents, where the grin1 knockout is embryonic lethal, grin1 double-mutant fish (grin1a -/- ; grin1b -/-), which lack all NMDAR-mediated synaptic transmission, survive until ∼10 d dpf (days post fertilization), providing a unique opportunity to explore NMDAR function during development and in generating behaviors. Many behavioral defects in the grin1 double-mutant larvae, including abnormal evoked responses to light and acoustic stimuli, prey-capture deficits, and a failure to habituate to acoustic stimuli, are replicated by short-term treatment with the NMDAR antagonist MK-801, suggesting that they arise from acute effects of compromised NMDAR-mediated transmission. Other defects, however, such as periods of hyperactivity and alterations in place preference, are not phenocopied by MK-801, suggesting a developmental origin. Together, we have developed a unique model to study NMDARs in the developing vertebrate nervous system.SIGNIFICANCE STATEMENT Rapid communication between cells in the nervous system depends on ion channels that are directly activated by chemical neurotransmitters. One such ligand-gated ion channel, the NMDAR, impacts nearly all forms of nervous system function. It has been challenging, however, to study the prolonged absence of NMDARs in vertebrates, and hence their role in nervous system development, due to experimental limitations. Here, we demonstrate that zebrafish lacking all NMDAR transmission are viable through early development and are capable of a wide range of stereotypic behaviors. As such, this zebrafish model provides a unique opportunity to study the role of NMDAR in the development of the early vertebrate nervous system.


Assuntos
Sistema Nervoso/embriologia , Sistema Nervoso/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Estimulação Acústica/métodos , Animais , Animais Geneticamente Modificados , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Células HEK293 , Humanos , Masculino , Sistema Nervoso/efeitos dos fármacos , Estimulação Luminosa/métodos , Ratos , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores
4.
Gene ; 744: 144632, 2020 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-32240777

RESUMO

IGF2BPs, a subclass of RNA-binding proteins, regulate cellular differentiation, proliferation and migration during multiple organs development, but their functions in liver development still remain unclear. Here, in this study, whole-mount in situ hybridization showed that igf2bp1 was constantly and stably expressed at early stages of embryo development in zebrafish. Both the morpholino-induced knockdown and CRISPR/Cas9-mediated knockout of igf2bp1 led to a reduced-size liver phenotype. Further analysis revealed that igf2bp1 is required for hepatic outgrowth, but not for hepatoblast specification and budding. Deficiency of igf2bp1 resulted in reduced cell proliferation, but had no effect on apoptosis. Therefore, we concluded that igf2bp1 is a critical factor to regulate hepatic outgrowth via cell proliferation during early liver development in zebrafish.


Assuntos
Fígado/embriologia , Proteínas de Ligação a RNA/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Animais , Proliferação de Células , Hepatócitos/citologia , Fígado/anatomia & histologia , Fígado/metabolismo , Morfolinos , Tamanho do Órgão , Fenótipo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
5.
Nat Commun ; 11(1): 479, 2020 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-31980602

RESUMO

Congenital scoliosis (CS) is a complex genetic disorder characterized by vertebral malformations. The precise etiology of CS is not fully defined. Here, we identify that mutation in dual serine/threonine and tyrosine protein kinase (dstyk) lead to CS-like vertebral malformations in zebrafish. We demonstrate that the scoliosis in dstyk mutants is related to the wavy and malformed notochord sheath formation and abnormal axial skeleton segmentation due to dysregulated biogenesis of notochord vacuoles and notochord function. Further studies show that DSTYK is located in late endosomal/lysosomal compartments and is involved in the lysosome biogenesis in mammalian cells. Dstyk knockdown inhibits notochord vacuole and lysosome biogenesis through mTORC1-dependent repression of TFEB nuclear translocation. Inhibition of mTORC1 activity can rescue the defect in notochord vacuole biogenesis and scoliosis in dstyk mutants. Together, our findings reveal a key role of DSTYK in notochord vacuole biogenesis, notochord morphogenesis and spine development through mTORC1/TFEB pathway.


Assuntos
Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Escoliose/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/anormalidades , Peixe-Zebra/genética , Transporte Ativo do Núcleo Celular , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Modelos Biológicos , Mutação , Notocorda/anormalidades , Notocorda/metabolismo , Notocorda/ultraestrutura , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Escoliose/congênito , Escoliose/metabolismo , Transdução de Sinais , Coluna Vertebral/anormalidades , Coluna Vertebral/metabolismo , Fatores de Transcrição/metabolismo , Vacúolos/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo
6.
Biochim Biophys Acta Mol Basis Dis ; 1866(5): 165662, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31917327

RESUMO

Pompe disease (PD) is an autosomal recessive muscular disorder caused by deficiency of the glycogen hydrolytic enzyme acid α-glucosidase (GAA). The enzyme replacement therapy, currently the only available therapy for PD patients, is efficacious in improving cardiomyopathy in the infantile form, but not equally effective in the late onset cases with involvement of skeletal muscle. Correction of the skeletal muscle phenotype has indeed been challenging, probably due to concomitant dysfunctional autophagy. The increasing attention to the pathogenic mechanisms of PD and the search of new therapeutic strategies prompted us to generate and characterize a novel transient PD model, using zebrafish. Our model presented increased glycogen content, markedly altered motor behavior and increased lysosome content, in addition to altered expression of the autophagy-related transcripts and proteins Beclin1, p62 and Lc3b. Furthermore, the model was used to assess the beneficial effects of 3-bromopyruvic acid (3-BrPA). Treatment with 3-BrPA induced amelioration of the model phenotypes regarding glycogen storage, motility behavior and autophagy-related transcripts and proteins. Our zebrafish PD model recapitulates most of the defects observed in human patients, proving to be a powerful translational model. Moreover, 3-BrPA unveiled to be a promising compound for treatment of conditions with glycogen accumulation.


Assuntos
Doença de Depósito de Glicogênio Tipo II/tratamento farmacológico , Glicogênio/metabolismo , Hexoquinase/antagonistas & inibidores , Piruvatos/farmacologia , Animais , Animais Geneticamente Modificados , Autofagia/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Técnicas de Silenciamento de Genes , Doença de Depósito de Glicogênio Tipo II/genética , Doença de Depósito de Glicogênio Tipo II/patologia , Glicólise/efeitos dos fármacos , Hexoquinase/metabolismo , Humanos , Lisossomos , Microscopia Eletrônica , Morfolinos/administração & dosagem , Morfolinos/genética , Atividade Motora/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Piruvatos/uso terapêutico , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , alfa-Glucosidases/genética , alfa-Glucosidases/metabolismo
7.
J Med Chem ; 63(1): 295-308, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31793776

RESUMO

Inhibition of histone deacetylase 6 (HDAC6) has emerged as a promising therapeutic strategy for the treatment of cancer, chemotherapy-induced peripheral neuropathy, and neurodegenerative disease. The recent X-ray crystal structure determination of HDAC6 enables an understanding of structural features directing affinity and selectivity in the active site. Here, we present the X-ray crystal structures of five HDAC6-inhibitor complexes that illuminate key molecular features of the inhibitor linker and capping groups that facilitate and differentiate binding to HDAC6. In particular, aromatic and heteroaromatic linkers nestle within an aromatic cleft defined by F583 and F643, and different aromatic linkers direct the capping group toward shallow pockets defined by the L1 loop, the L2 loop, or somewhere in between these pockets. These results expand our understanding of factors contributing to the selective inhibition of HDAC6, particularly regarding interactions that can be targeted in the region of the L2 pocket.


Assuntos
Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo , Animais , Domínio Catalítico , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Interações Hidrofóbicas e Hidrofílicas , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Peixe-Zebra
8.
J Leukoc Biol ; 107(2): 185-203, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31529512

RESUMO

The CXCR3-CXCL11 chemokine-signaling axis plays an essential role in infection and inflammation by orchestrating leukocyte trafficking in human and animal models, including zebrafish. Atypical chemokine receptors (ACKRs) play a fundamental regulatory function in signaling networks by shaping chemokine gradients through their ligand scavenging function, while being unable to signal in the classic G-protein-dependent manner. Two copies of the CXCR3 gene in zebrafish, cxcr3.2 and cxcr3.3, are expressed on macrophages and share a highly conserved ligand-binding site. However, Cxcr3.3 has structural characteristics of ACKRs indicative of a ligand-scavenging role. In contrast, we previously showed that Cxcr3.2 is an active CXCR3 receptor because it is required for macrophage motility and recruitment to sites of mycobacterial infection. In this study, we generated a cxcr3.3 CRISPR-mutant to functionally dissect the antagonistic interplay among the cxcr3 paralogs in the immune response. We observed that cxcr3.3 mutants are more susceptible to mycobacterial infection, whereas cxcr3.2 mutants are more resistant. Furthermore, macrophages in the cxcr3.3 mutant are more motile, show higher activation status, and are recruited more efficiently to sites of infection or injury. Our results suggest that Cxcr3.3 is an ACKR that regulates the activity of Cxcr3.2 by scavenging common ligands and that silencing the scavenging function of Cxcr3.3 results in an exacerbated Cxcr3.2 signaling. In human, splice variants of CXCR3 have antagonistic functions and CXCR3 ligands also interact with ACKRs. Therefore, in zebrafish, an analogous regulatory mechanism appears to have evolved after the cxcr3 gene duplication event, through diversification of conventional and atypical receptor variants.


Assuntos
Movimento Celular , Macrófagos/fisiologia , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium marinum/fisiologia , Receptores CXCR3/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Sistemas CRISPR-Cas , Macrófagos/citologia , Macrófagos/microbiologia , Mutação , Infecções por Mycobacterium não Tuberculosas/metabolismo , Infecções por Mycobacterium não Tuberculosas/patologia , Conformação Proteica , Receptores CXCR3/antagonistas & inibidores , Receptores CXCR3/classificação , Receptores CXCR3/genética , Peixe-Zebra/microbiologia , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética
9.
Int J Mol Sci ; 21(1)2019 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-31881740

RESUMO

The G protein-coupled cannabinoid receptors type 1 (CB1R) and type 2 (CB2R), and their endocannabinoid (eCBs) ligands, have been implicated in several aspects of brain wiring during development. Here we aim to assess whether interfering with CB1R affects development, neuritogenesis and pathfinding of GnRH and AgRP neurons, forebrain neurons that control respectively reproduction and appetite. We pharmacologically and genetically interfered with CB1R in zebrafish strains with fluorescently labeled GnRH3 and the AgRP1 neurons. By applying CB1R antagonists we observed a reduced number of GnRH3 neurons, fiber misrouting and altered fasciculation. Similar phenotypes were observed by CB1R knockdown. Interfering with CB1R also resulted in a reduced number, misrouting and poor fasciculation of the AgRP1 neuron's axonal projections. Using a bioinformatic approach followed by qPCR validation, we have attempted to link CB1R functions with known guidance and fasciculation proteins. The search identified stathmin-2, a protein controlling microtubule dynamics, previously demonstrated to be coexpressed with CB1R and now shown to be downregulated upon interference with CB1R in zebrafish. Together, these results raise the likely possibility that embryonic exposure to low doses of CB1R-interfering compounds could impact on the development of the neuroendocrine systems controlling sexual maturation, reproduction and food intake.


Assuntos
Proteína Relacionada com Agouti/metabolismo , Axônios/metabolismo , Hormônio Liberador de Gonadotropina/metabolismo , Ácido Pirrolidonocarboxílico/análogos & derivados , Receptor CB1 de Canabinoide/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Benzoxazinas/farmacologia , Embrião não Mamífero/efeitos dos fármacos , Embrião não Mamífero/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Morfolinas/farmacologia , Morfolinos/metabolismo , Naftalenos/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ácido Pirrolidonocarboxílico/metabolismo , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética
10.
Methods Enzymol ; 626: 447-474, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31606087

RESUMO

Histone deacetylase (HDAC) isozymes modulate numerous regulatory signals and pathways in biological systems, hence serving as targets for drug design. For example, HDAC6 is the cytosolic tubulin deacetylase and its inhibition compromises microtubule dynamics, leading to cancer cell cycle arrest and apoptosis. The design of inhibitors that selectively target HDAC6 is desirable to avoid side effects resulting from the inhibition of off-target HDACs. High resolution X-ray crystal structures of HDAC6 have accelerated structure-based approaches to drug design targeting HDAC6. Crystal structure analysis reveals that the tubulin deacetylase domain of human HDAC6 (catalytic domain 2, also known as CD2) is very similar to that of HDAC6 CD2 from Danio rerio (zebrafish, designated zCD2). Thus, zCD2 is a valid surrogate of human HDAC6 CD2, the actual drug target; moreover, zCD2 is much more easily prepared and crystallized. A plasmid containing the zCD2 construct for heterologous expression in Escherichia coli is available through Addgene (#122031). In this chapter, we review the preparation, purification, and crystallization of zCD2-inhibitor complexes. These methods enable the rapid acquisition of structural data regarding optimal zinc-binding groups, capping groups, and linkers in the discovery of new and selective HDAC6 inhibitors.


Assuntos
Desenho de Fármacos , Desacetilase 6 de Histona/química , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Proteínas de Peixe-Zebra/química , Peixe-Zebra , Animais , Sítios de Ligação/efeitos dos fármacos , Cristalização/métodos , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/metabolismo , Humanos , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo , Zinco/metabolismo
11.
J Cell Sci ; 132(22)2019 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-31636113

RESUMO

Osteoblast differentiation is a key process for bone homeostasis and repair. Multiple signalling pathways have been associated with osteoblast differentiation, yet much remains unknown on how this process is regulated in vivo Previous studies have proposed that the Hippo pathway transcriptional co-activators YAP and TAZ (also known as YAP1 and WWTR1, respectively) maintain progenitor stemness and inhibit terminal differentiation of osteoblasts, whereas others suggest they potentiate osteoblast differentiation and bone formation. Here, we use zebrafish caudal fin regeneration as a model to clarify how the Hippo pathway regulates de novo bone formation and osteoblast differentiation. We demonstrate that Yap inhibition leads to accumulation of osteoprogenitors and prevents osteoblast differentiation in a cell non-autonomous manner. This effect correlates with a severe impairment of Bmp signalling in osteoblasts, likely by suppressing the expression of the ligand bmp2a in the surrounding mesenchymal cells. Overall, our findings provide a new mechanism of bone formation through the Hippo-Yap pathway, integrating Yap in the signalling cascade that governs osteoprogenitor maintenance and subsequent differentiation during zebrafish caudal fin regeneration.


Assuntos
Nadadeiras de Animais/fisiologia , Proteínas Morfogenéticas Ósseas/genética , Osteoblastos/metabolismo , Regeneração/fisiologia , Transativadores/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Nadadeiras de Animais/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células , Osteoblastos/citologia , Osteogênese , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Transativadores/antagonistas & inibidores , Proteínas de Peixe-Zebra/antagonistas & inibidores
12.
Elife ; 82019 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-31609202

RESUMO

Sensory hair cells in the ear utilize specialized ribbon synapses. These synapses are defined by electron-dense presynaptic structures called ribbons, composed primarily of the structural protein Ribeye. Previous work has shown that voltage-gated influx of Ca2+ through CaV1.3 channels is critical for hair-cell synapse function and can impede ribbon formation. We show that in mature zebrafish hair cells, evoked presynaptic-Ca2+ influx through CaV1.3 channels initiates mitochondrial-Ca2+ (mito-Ca2+) uptake adjacent to ribbons. Block of mito-Ca2+ uptake in mature cells depresses presynaptic-Ca2+ influx and impacts synapse integrity. In developing zebrafish hair cells, mito-Ca2+ uptake coincides with spontaneous rises in presynaptic-Ca2+ influx. Spontaneous mito-Ca2+ loading lowers cellular NAD+/NADH redox and downregulates ribbon size. Direct application of NAD+ or NADH increases or decreases ribbon size respectively, possibly acting through the NAD(H)-binding domain on Ribeye. Our results present a mechanism where presynaptic- and mito-Ca2+ couple to confer proper presynaptic function and formation.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo , Potenciais Evocados Auditivos/fisiologia , Proteínas do Olho/metabolismo , Células Ciliadas Auditivas/metabolismo , Mitocôndrias/metabolismo , Sinapses/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Animais , Animais Geneticamente Modificados , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/genética , Sinalização do Cálcio , Tamanho Celular , Embrião não Mamífero , Proteínas do Olho/química , Proteínas do Olho/genética , Expressão Gênica , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/efeitos dos fármacos , Isradipino/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , NAD/metabolismo , Oxirredução , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Compostos de Rutênio/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/ultraestrutura , Transmissão Sináptica , Peixe-Zebra , Proteínas de Peixe-Zebra/agonistas , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética
13.
Sci Rep ; 9(1): 12405, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31455847

RESUMO

The circadian clock ensures that behavioral and physiological processes occur at appropriate times during the 24-hour day/night cycle, and is regulated at both the cellular and organismal levels. To identify pathways acting on intact animals, we performed a small molecule screen using a luminescent reporter of molecular circadian rhythms in zebrafish larvae. We identified both known and novel pathways that affect circadian period, amplitude and phase. Several drugs identified in the screen did not affect circadian rhythms in cultured cells derived from luminescent reporter embryos or in established zebrafish and mammalian cell lines, suggesting they act via mechanisms absent in cell culture. Strikingly, using drugs that promote or inhibit inflammation, as well as a mutant that lacks microglia, we found that inflammatory state affects circadian amplitude. These results demonstrate a benefit of performing drug screens using intact animals and provide novel targets for treating circadian rhythm disorders.


Assuntos
Ritmo Circadiano/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados/fisiologia , Anti-Inflamatórios não Esteroides/farmacologia , Caseína Quinase I/antagonistas & inibidores , Caseína Quinase I/metabolismo , Larva/efeitos dos fármacos , Larva/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Receptores da Glicina/agonistas , Receptores da Glicina/metabolismo , Taurina/farmacologia , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo
14.
Bioorg Med Chem ; 27(19): 115014, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31358358

RESUMO

A new series of 1,3-diketone, heterocyclic and α,ß-unsaturated derivatives were synthesized and evaluated for their AhR antagonist activity using zebrafish and mammalian cells. Compounds 1b, 2c, 3b and 5b showed significant AhR antagonist activity in a transgenic zebrafish model. Among them, compound 3b, and 5b were found to have excellent AhR antagonist activity with IC50 of 3.36 nM and 8.3 nM in a luciferase reporter gene assay. In stem cell proliferation assay, compound 5b elicited marked HSC expansion.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/antagonistas & inibidores , Chalconas/farmacologia , Receptores de Hidrocarboneto Arílico/antagonistas & inibidores , Proteínas de Peixe-Zebra/antagonistas & inibidores , Animais , Células COS , Proliferação de Células/efeitos dos fármacos , Chalconas/síntese química , Chlorocebus aethiops , Humanos , Células-Tronco/efeitos dos fármacos , Peixe-Zebra
15.
FASEB J ; 33(10): 10648-10667, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31268746

RESUMO

Casein kinase 2 (CK2) is a tetrameric protein kinase composed of 2 catalytic (α and α') and 2 regulatory ß subunits. Our study provides the first molecular and cellular characterization of the different CK2 subunits, highlighting their individual roles in skeletal muscle specification and differentiation. Analysis of C2C12 cell knockout for each CK2 subunit reveals that: 1) CK2ß is mandatory for the expression of the muscle master regulator myogenic differentiation 1 in proliferating myoblasts, thus controlling both myogenic commitment and subsequent muscle-specific gene expression and myotube formation; 2) CK2α is involved in the activation of the muscle-specific gene program; and 3) CK2α' activity regulates myoblast fusion by mediating plasma membrane translocation of fusogenic proteins essential for membrane coalescence, like myomixer. Accordingly, CK2α' overexpression in C2C12 cells and in mouse regenerating muscle is sufficient to increase myofiber size and myonuclei content via enhanced satellite cell fusion. Consistent with these results, pharmacological inhibition of CK2 activity substantially blocks the expression of myogenic markers and muscle cell fusion both in vitro in C2C12 and primary myoblasts and in vivo in mouse regenerating muscle and zebrafish development. Overall, our work describes the specific and coordinated functions of CK2 subunits in orchestrating muscle differentiation and fusogenic activity, highlighting CK2 relevance in the physiopathology of skeletal muscle tissue.-Salizzato, V., Zanin, S., Borgo, C., Lidron, E., Salvi, M., Rizzuto, R., Pallafacchina, G., Donella-Deana, A. Protein kinase CK2 subunits exert specific and coordinated functions in skeletal muscle differentiation and fusogenic activity.


Assuntos
Caseína Quinase II/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/enzimologia , Animais , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/genética , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Fusão Celular , Linhagem Celular , Técnicas de Inativação de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia , Proteína MyoD/genética , Proteína MyoD/metabolismo , Mioblastos Esqueléticos/citologia , Mioblastos Esqueléticos/enzimologia , Subunidades Proteicas , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/enzimologia , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/fisiologia
16.
Artigo em Inglês | MEDLINE | ID: mdl-31255699

RESUMO

Organic anion transporters (OATs) are membrane proteins within the Solute carrier family 22 (SLC22). They play important roles in cellular uptake of various organic compounds, and due to their expression in barrier tissues of major excretory and non-excretory organs are considered as crucial elements in absorption and distribution of a wide range of endobiotic and xenobiotic compounds. Based on our previous work and initial insights on SLC22 members in zebrafish (Danio rerio), in this study we aimed at in vitro characterization of Oat1 and Oat3 transporters and understanding of their interaction with potential physiological substrates. We first performed synteny analysis to describe in more detail orthological relationship of zebrafish oat1 and oat3 genes. We then developed stable cell lines overexpressing Oat1 and Oat3, and identified Lucifer yellow as Oat1 model fluorescent substrate (Km = 11.4 µM) and 6-carboxyfluorescein as Oat3 model substrate (Km = 5.8 µM). Initial identification performed using the developed assays revealed Kreb's cycle intermediates, bilirubin, bile salts and steroid hormones as the most potent of Oat1 and Oat3 interactors, with IC50 values in micromolar range. Finally, we showed that bilirubin, deoxycholic acid, α-ketoglutarate, pregnenolone, estrone-3-sulfate and corticosterone are in vitro substrates of zebrafish Oat1, and bilirubin and deoxycholic acid are Oat3 substrates. In conclusion, using the approach described, structural and functional similarities of both transporters to human and mammalian orthologs are revealed, their broad ligand selectivity confirmed, potent interactors among endobiotic compounds identified, and first indications of their potential physiological role(s) in zebrafish obtained.


Assuntos
Proteína 1 Transportadora de Ânions Orgânicos/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos , Proteína 1 Transportadora de Ânions Orgânicos/antagonistas & inibidores , Transportadores de Ânions Orgânicos Sódio-Independentes/antagonistas & inibidores , Ligação Proteica , Transporte Proteico , Proteínas de Peixe-Zebra/antagonistas & inibidores
17.
AIDS ; 33(13): 1967-1976, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31259764

RESUMO

OBJECTIVE: Maternal folate (vitamin B9) status is the largest known modifier of neural tube defect risk, so we evaluated folate-related mechanisms of action for dolutegravir (DTG) developmental toxicity. DESIGN: Folate receptor 1 (FOLR1) was examined as a target for DTG developmental toxicity using protein and cellular interaction studies and an animal model. METHODS: FOLR1 competitive binding studies were used to test DTG for FOLR1 antagonism. Human placenta cell line studies were used to test interactions with DTG, folate, and cations. Zebrafish were selected as an animal model to examine DTG-induced developmental toxicity and rescue strategies. RESULTS: FOLR1 binding studies indicate DTG is a noncompetitive FOLR1 antagonist at therapeutic concentrations. In-vitro testing indicates calcium (2 mmol/l) increases FOLR1-folate interactions and alters DTG-FOLR1-folate interactions and cytotoxicity. DTG does not inhibit downstream folate metabolism by dihydrofolate reductase. Early embryonic exposure to DTG is developmentally toxic in zebrafish, and supplemental folic acid can mitigate DTG developmental toxicity. CONCLUSION: Folates and FOLR1 are established modifiers of risk for neural tube defects, and binding data indicates DTG is a partial antagonist of FOLR1. Supplemental folate can ameliorate increased developmental toxicity due to DTG in zebrafish. The results from these studies are expected to inform and guide future animal models and clinical studies of DTG-based antiretroviral therapy in women of childbearing age.


Assuntos
Receptor 1 de Folato/antagonistas & inibidores , Ácido Fólico/administração & dosagem , Compostos Heterocíclicos com 3 Anéis/toxicidade , Proteínas de Peixe-Zebra/antagonistas & inibidores , Peixe-Zebra/embriologia , Animais , Linhagem Celular , Suplementos Nutricionais , Desenvolvimento Embrionário/efeitos dos fármacos , Feminino , Receptor 1 de Folato/genética , Infecções por HIV/tratamento farmacológico , Inibidores de Integrase de HIV/toxicidade , Humanos , Modelos Animais , Oxazinas , Piperazinas , Gravidez , Piridonas , Testes de Toxicidade , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
18.
J Biol Chem ; 294(31): 11741-11750, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31186352

RESUMO

The Notch receptor is a key mediator of developmental programs and cell-fate decisions. Imbalanced Notch signaling leads to developmental disorders and cancer. To fully characterize the Notch signaling pathway and exploit it in novel therapeutic interventions, a comprehensive view on the regulation and requirements of Notch signaling is needed. Notch is regulated at different levels, ranging from ligand binding, stability to endocytosis. Using an array of different techniques, including reporter gene assays, immunocytochemistry, and ChIP-qPCR we show here, to the best of our knowledge for the first time, regulation of Notch signaling at the level of the nuclear pore. We found that the nuclear pore protein Nup214 (nucleoporin 214) and its interaction partner Nup88 negatively regulate Notch signaling in vitro and in vivo in zebrafish. In mammalian cells, loss of Nup88/214 inhibited nuclear export of recombination signal-binding protein for immunoglobulin κJ region (RBP-J), the DNA-binding component of the Notch pathway. This inhibition increased binding of RBP-J to its cognate promoter regions, resulting in increased downstream Notch signaling. Interestingly, we also found that NUP214 fusion proteins, causative for certain cases of T-cell acute lymphatic leukemia, potentially contribute to tumorigenesis via a Notch-dependent mechanism. In summary, the nuclear pore components Nup88/214 suppress Notch signaling in vitro, and in zebrafish, nuclear RBP-J levels are rate-limiting factors for Notch signaling in mammalian cells, and regulation of nucleocytoplasmic transport of RBP-J may contribute to fine-tuning Notch activity in cells.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Transdução de Sinais , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular Tumoral , Humanos , Proteína de Ligação a Sequências Sinal de Recombinação J de Imunoglobina/metabolismo , Morfolinos/genética , Morfolinos/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/antagonistas & inibidores , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Receptores Notch/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição HES-1/antagonistas & inibidores , Fatores de Transcrição HES-1/genética , Fatores de Transcrição HES-1/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
19.
Biochem Biophys Res Commun ; 516(3): 680-685, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31248596

RESUMO

Acute renal injury (AKI) is a serious disorder of renal failure or renal damage that occurs within hours or days. At present, there is no approved pharmaceutical treatment for AKI. Zebrafish is an excellent model for studying the repair of AKI because of its remarkable ability to repair kidney injury. Using zebrafish AKI model inducing by gentamicin, we found that hydrogen peroxide (H2O2) plays dual roles during the period of AKI recovery including renal repair and kidney regeneration. In the repair stage of AKI, H2O2 was produced in proximal and distal segments of renal tubules. By inhibiting H2O2 generation using Duox Vivo-Morpholino or chemical inhibitor, it was observed of severe damage of renal tubules, and extensive cell apoptosis. In the stage of regeneration, we found that H2O2 was highly generated in renal interstitium. Inhibiting production of H2O2 could significantly down-regulate the ability of kidney regeneration, which was associated with the failure of proliferation of renal progenitor cells. Therefore, H2O2 acts as a protective factor in renal repair and an initial signal of kidney regeneration, indicating the key roles of H2O2 in promoting recovery of AKI in zebrafish.


Assuntos
Lesão Renal Aguda/metabolismo , Modelos Animais de Doenças , Peróxido de Hidrogênio/metabolismo , Túbulos Renais Proximais/metabolismo , Transdução de Sinais/fisiologia , Lesão Renal Aguda/genética , Animais , Apoptose/fisiologia , Proliferação de Células/fisiologia , Humanos , Peróxido de Hidrogênio/antagonistas & inibidores , Rim/metabolismo , Rim/patologia , Rim/fisiopatologia , Túbulos Renais Proximais/patologia , Morfolinos , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Regeneração/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
20.
Dis Model Mech ; 12(5)2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31043432

RESUMO

DYRK1A is a major causative gene in Down syndrome (DS). Reduced incidence of solid tumors such as neuroblastoma in DS patients and increased vascular anomalies in DS fetuses suggest a potential role of DYRK1A in angiogenic processes, but in vivo evidence is still scarce. Here, we used zebrafish dyrk1aa mutant embryos to understand DYRK1A function in cerebral vasculature formation. Zebrafish dyrk1aa mutants exhibited cerebral hemorrhage and defects in angiogenesis of central arteries in the developing hindbrain. Such phenotypes were rescued by wild-type dyrk1aa mRNA, but not by a kinase-dead form, indicating the importance of DYRK1A kinase activity. Chemical screening using a bioactive small molecule library identified a calcium chelator, EGTA, as one of the hits that most robustly rescued the hemorrhage. Vascular defects of mutants were also rescued by independent modulation of calcium signaling by FK506. Furthermore, the transcriptomic analyses supported the alterations of calcium signaling networks in dyrk1aa mutants. Together, our results suggest that DYRK1A plays an essential role in angiogenesis and in maintenance of the developing cerebral vasculature via regulation of calcium signaling, which may have therapeutic potential for DYRK1A-related vascular diseases.


Assuntos
Vasos Sanguíneos/patologia , Sinalização do Cálcio , Técnicas de Inativação de Genes , Proteínas Quinases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Vasos Sanguíneos/efeitos dos fármacos , Encéfalo/irrigação sanguínea , Encéfalo/embriologia , Encéfalo/patologia , Encéfalo/ultraestrutura , Hemorragia Cerebral/patologia , Ácido Egtázico/farmacologia , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Desenvolvimento Embrionário/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Harmina/farmacologia , Espaço Intracelular/metabolismo , Mutação/genética , Fenótipo , Transcriptoma/genética , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/antagonistas & inibidores
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