RESUMO
The characteristics of the sleep drivers and the mechanisms through which sleep relieves the cellular homeostatic pressure are unclear. In flies, zebrafish, mice, and humans, DNA damage levels increase during wakefulness and decrease during sleep. Here, we show that 6 h of consolidated sleep is sufficient to reduce DNA damage in the zebrafish dorsal pallium. Induction of DNA damage by neuronal activity and mutagens triggered sleep and DNA repair. The activity of the DNA damage response (DDR) proteins Rad52 and Ku80 increased during sleep, and chromosome dynamics enhanced Rad52 activity. The activity of the DDR initiator poly(ADP-ribose) polymerase 1 (Parp1) increased following sleep deprivation. In both larva zebrafish and adult mice, Parp1 promoted sleep. Inhibition of Parp1 activity reduced sleep-dependent chromosome dynamics and repair. These results demonstrate that DNA damage is a homeostatic driver for sleep, and Parp1 pathways can sense this cellular pressure and facilitate sleep and repair activity.
Assuntos
Comportamento Animal , Encéfalo , Dano ao DNA , Reparo do DNA , Neurônios , Poli(ADP-Ribose) Polimerase-1 , Sono , Proteínas de Peixe-Zebra , Animais , Feminino , Masculino , Animais Geneticamente Modificados , Encéfalo/enzimologia , Encéfalo/patologia , Encéfalo/fisiopatologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Camundongos Endogâmicos C57BL , Neurônios/enzimologia , Neurônios/patologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/fisiologia , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Fatores de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
Brain nuclei are traditionally defined by their anatomy, activity, and expression of specific markers. The hypothalamus contains discrete neuronal populations that coordinate fundamental behavioral functions, including sleep and wakefulness, in all vertebrates. Particularly, the diverse roles of hypocretin/orexin (Hcrt)-releasing neurons suggest functional heterogeneity among Hcrt neurons. Using single-cell RNA sequencing (scRNA-seq) and high-resolution imaging of the adult male and female zebrafish hypothalamic periventricular zone (PVZ), we identified 21 glutamatergic and 28 GABAergic cell types. Integration of zebrafish and mouse scRNA-seq revealed evolutionary conserved and divergent hypothalamic cell types. The expression of specific genes, including npvf, which encodes a sleep-regulating neuropeptide, was enriched in subsets of glutamatergic Hcrt neurons in both larval and adult zebrafish. The genetic profile, activity, and neurite processing of the neuronal subpopulation that co-expresses both Hcrt and Npvf (Hcrt+Npvf+) differ from other Hcrt neurons. These inter-species findings provide a unified annotation of hypothalamic cell types, and suggest that the heterogeneity of Hcrt neurons enables multi-functionality, such as consolidation of both wake and sleep by the Hcrt- and Npvf-releasing neuronal subpopulation.Significance Statement The study reveals the intricate heterogeneity within the hypothalamic periventricular zone (PVZ) of zebrafish, identifying 21 glutamatergic and 28 GABAergic cell types through single-cell RNA sequencing (scRNA-seq) and high-resolution imaging. Comparative analysis with mouse scRNA-seq data revealed conserved and divergent cell types, transcriptional regulatory mechanisms, and neuropeptide localization. Notably, we identified a unique neuronal subpopulation co-expressing both hypocretin/orexin (Hcrt) and neuropeptide VF (Npvf) neuropeptides in zebrafish. The distinct genetic profiles, activity patterns, and neurite processing of this subpopulation suggest a role in regulating both sleep and wakefulness.
RESUMO
Thyroid hormones (THs), thyroxine (T4), and triiodothyronine (T3), regulate growth, metabolism, and neurodevelopment. THs secretion is controlled by the pituitary thyroid-stimulating hormone (TSH) and the hypothalamic-pituitary-thyroid (HPT) axis. The organic anion-transporting polypeptide 1C1 (OATP1C1/SLCO1C1) and the monocarboxylate transporter 8 (MCT8/SLC16A2) actively transport THs, which bind to their nuclear receptors and induce gene expression. A mutation in OATP1C1 is associated with brain hypometabolism, gradual neurodegeneration, and impaired cognitive and motor functioning in adolescent patients. To understand the role of Oatp1c1 and the mechanisms of the disease, we profiled the transcriptome of oatp1c1 mutant (oatp1c1 -/-) and mct8 -/- xoatp1c1 -/- adult male and female zebrafish brains. Among dozens of differentially expressed genes, agouti-related neuropeptide 1 (agrp1) expression increased in oatp1c1 -/- adult brains. Imaging in the hypothalamus revealed enhanced proliferation of Agrp1 neurons in oatp1c1 -/- larvae and adults, and increased food consumption in oatp1c1 -/- larvae. Similarly, feeding and the number of Agrp1 neurons increased in thyroid gland-ablated zebrafish. Pharmacological treatments showed that the T3 analog TRIAC (3,3',5-tri-iodothyroacetic acid), but not T4, normalized the number of Agrp1 neurons in oatp1c1 -/- zebrafish. Since the HPT axis is hyperactive in the oatp1c1 -/- brain, we used the CRISPR-Cas9 system to knockdown tsh in oatp1c1 -/- larvae, and inducibly enhanced the HPT axis in wild-type larvae. These manipulations showed that Tsh promotes proliferation of Agrp1 neurons and increases food consumption in zebrafish. The results revealed upregulation of both the HPT axis-Agrp1 circuitry and feeding in a zebrafish model for OATP1C1 deficiency.SIGNIFICANCE STATEMENT Mutation in the thyroid hormone (TH) transporter OATP1C1 is associated with cognitive and motor functioning disturbances in humans. Here, we used an oatp1c1 -/- zebrafish to understand the role of organic anion-transporting polypeptide 1C1 (Oatp1c1), and the characteristics of OATP1C1 deficiency. Transcriptome profiling identified upregulation of agrp1 expression in the oatp1c1 -/- brain. The oatp1c1 -/- larvae showed increased thyroid-stimulating hormone (tsh) levels, proliferation of Agrp1 neurons and food consumption. Genetic manipulations of the hypothalamic-pituitary-thyroid (HPT) axis showed that Tsh increases the number of Agrp1 neurons and food consumption. The T3 analog TRIAC (3,3',5-tri-iodothyroacetic acid) normalizes the number of Agrp1 neurons and may have potential for the treatment of Oatp1c1 deficiency. The findings demonstrate a functional interaction between the thyroid and feeding systems in the brain of zebrafish and suggest a neuroendocrinological mechanism for OATP1C1 deficiency.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Transportadores de Ácidos Monocarboxílicos , Transportadores de Ânions Orgânicos , Simportadores , Adolescente , Animais , Feminino , Humanos , Masculino , Ânions , Proliferação de Células , Larva/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Neurônios/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Simportadores/metabolismo , Hormônios Tireóideos , Tireotropina/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismoRESUMO
Thyroid hormones (THs; T3 and T4) enter cells using specific transporters and regulate development and metabolism. Mutation in the TH transporter monocarboxylate transporter 8 (MCT8, SLC16A2) is associated with brain hypothyroidism and neurological impairment. We established mct8 mutant (mct8-/-) zebrafish as a model for MCT8 deficiency, which causes endocrinological, neurological, and behavioral alterations. Here, we profiled the transcriptome of mct8-/- larvae. Among hundreds of differentially expressed genes, the expression of a cluster of vision-related genes was distinct. Specifically, the expression of the opsin 1 medium wave sensitive 2 (opn1mw2) decreased in two mct8 mutants: mct8-/- and mct8-25bp-/- larvae, and under pharmacological inhibition of TH production. Optokinetic reflex (OKR) assays showed a reduction in the number of conjugated eye movements, and live imaging of genetically encoded Ca2+ indicator revealed altered neuronal activity in the pretectum area of mct8-25bp-/- larvae. These results imply that MCT8 and THs regulate the development of the visual system and suggest a mechanism to the deficiencies observed in the visual system of MCT8-deficiency patients.
Assuntos
Hipotireoidismo , Simportadores , Animais , Encéfalo/metabolismo , Humanos , Hipotireoidismo/metabolismo , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Simportadores/genética , Simportadores/metabolismo , Hormônios Tireóideos/genética , Hormônios Tireóideos/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Hypothalamic neurotensin (Nts)-secreting neurons regulate fundamental physiological processes including metabolism and feeding. However, the role of Nts in modulation of locomotor activity, sleep, and arousal is unclear. We previously identified and characterized Nts neurons in the zebrafish hypothalamus. MATERIALS AND METHODS: In order to study the role of Nts, nts mutant (nts-/-), and overexpressing zebrafish were generated. RESULTS: The expression of both nts mRNA and Nts protein was reduced during the night in wild-type zebrafish. Behavioral assays revealed that locomotor activity was decreased during both day and night, while sleep was increased exclusively during the nighttime in nts-/- larvae. Likewise, inducible overexpression of Nts increased arousal in hsp70:Gal4/uas:Nts larvae. Furthermore, the behavioral response to light-to-dark transitions was reduced in nts-/- larvae. In order to elucidate potential contenders that may mediate Nts action on these behaviors, we profiled the transcriptome of 6 dpf nts-/- larvae. Among other genes, the expression levels of melanin-concentrating hormone receptor 1b were increased in nts-/- larvae. Furthermore, a portion of promelanin-concentrating hormone 1 (pmch1) and pmch2 neurons expressed the nts receptor. In addition, expression of the the two zebrafish melanin-concentrating hormone (Mch) orthologs, Mch1 and Mch2, was increased in nts-/- larvae. CONCLUSION: These results show that the Nts and Mch systems interact and modulate locomotor activity and arousal.
Assuntos
Nível de Alerta/fisiologia , Hormônios Hipotalâmicos/metabolismo , Locomoção/fisiologia , Melaninas/metabolismo , Neurotensina/fisiologia , Hormônios Hipofisários/metabolismo , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/metabolismo , Animais , Neurotensina/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
Fragile X syndrome (FXS) is the most frequent inherited form of mental retardation. The cause for this X-linked disorder is the silencing of the fragile X mental retardation 1 (fmr1) gene and the absence of the fragile X mental retardation protein (Fmrp). The RNA-binding protein Fmrp represses protein translation, particularly in synapses. In Drosophila, Fmrp interacts with the adenosine deaminase acting on RNA (Adar) enzymes. Adar enzymes convert adenosine to inosine (A-to-I) and modify the sequence of RNA transcripts. Utilizing the fmr1 zebrafish mutant (fmr1-/-), we studied Fmrp-dependent neuronal circuit formation, behavior, and Adar-mediated RNA editing. By combining behavior analyses and live imaging of single axons and synapses, we showed hyperlocomotor activity, as well as increased axonal branching and synaptic density, in fmr1-/- larvae. We identified thousands of clustered RNA editing sites in the zebrafish transcriptome and showed that Fmrp biochemically interacts with the Adar2a protein. The expression levels of the adar genes and Adar2 protein increased in fmr1-/- zebrafish. Microfluidic-based multiplex PCR coupled with deep sequencing showed a mild increase in A-to-I RNA editing levels in evolutionarily conserved neuronal and synaptic Adar-targets in fmr1-/- larvae. These findings suggest that loss of Fmrp results in increased Adar-mediated RNA editing activity on target-specific RNAs, which, in turn, might alter neuronal circuit formation and behavior in FXS.
Assuntos
Adenosina Desaminase/genética , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Proteínas de Ligação a RNA/genética , Proteínas de Peixe-Zebra/genética , Adenosina Desaminase/biossíntese , Animais , Axônios/metabolismo , Axônios/patologia , Modelos Animais de Doenças , Proteína do X Frágil da Deficiência Intelectual/biossíntese , Síndrome do Cromossomo X Frágil/patologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Atividade Motora/genética , Neurônios/metabolismo , Neurônios/patologia , Edição de RNA/genética , Proteínas de Ligação a RNA/biossíntese , Sinapses/metabolismo , Sinapses/patologia , Transcriptoma/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/biossínteseRESUMO
The mechanisms and treatment of psychomotor retardation, which includes motor and cognitive impairment, are indefinite. The Allan-Herndon-Dudley syndrome (AHDS) is an X-linked psychomotor retardation characterized by delayed development, severe intellectual disability, muscle hypotonia, and spastic paraplegia, in combination with disturbed thyroid hormone (TH) parameters. AHDS has been associated with mutations in the monocarboxylate transporter 8 (mct8/slc16a2) gene, which is a TH transporter. In order to determine the pathophysiological mechanisms of AHDS, MCT8 knockout mice were intensively studied. Although these mice faithfully replicated the abnormal serum TH levels, they failed to exhibit the neurological and behavioral symptoms of AHDS patients. Here, we generated an mct8 mutant (mct8-/-) zebrafish using zinc-finger nuclease (ZFN)-mediated targeted gene editing system. The elimination of MCT8 decreased the expression levels of TH receptors; however, it did not affect the expression of other TH-related genes. Similar to human patients, mct8-/- larvae exhibited neurological and behavioral deficiencies. High-throughput behavioral assays demonstrated that mct8-/- larvae exhibited reduced locomotor activity, altered response to external light and dark transitions and an increase in sleep time. These deficiencies in behavioral performance were associated with altered expression of myelin-related genes and neuron-specific deficiencies in circuit formation. Time-lapse imaging of single-axon arbors and synapses in live mct8-/- larvae revealed a reduction in filopodia dynamics and axon branching in sensory neurons and decreased synaptic density in motor neurons. These phenotypes enable assessment of the therapeutic potential of three TH analogs that can enter the cells in the absence of MCT8. The TH analogs restored the myelin and axon outgrowth deficiencies in mct8-/- larvae. These findings suggest a mechanism by which MCT8 regulates neural circuit assembly, ultimately mediating sensory and motor control of behavioral performance. We also propose that the administration of TH analogs early during embryo development can specifically reduce neurological damage in AHDS patients.
Assuntos
Deficiência Intelectual Ligada ao Cromossomo X/diagnóstico , Deficiência Intelectual Ligada ao Cromossomo X/etiologia , Hipotonia Muscular/diagnóstico , Hipotonia Muscular/etiologia , Atrofia Muscular/diagnóstico , Atrofia Muscular/etiologia , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Ordem dos Genes , Marcação de Genes , Sistema Hipotálamo-Hipofisário , Fatores de Transcrição Kruppel-Like/genética , Deficiência Intelectual Ligada ao Cromossomo X/tratamento farmacológico , Transportadores de Ácidos Monocarboxílicos/genética , Atividade Motora/genética , Hipotonia Muscular/tratamento farmacológico , Atrofia Muscular/tratamento farmacológico , Mutação , Bainha de Mielina/metabolismo , Neurogranina/genética , Neurônios/metabolismo , Fenótipo , Fotoperíodo , Pseudópodes/genética , Pseudópodes/metabolismo , Glândula Tireoide , Receptores alfa dos Hormônios Tireóideos/genética , Hormônios Tireóideos/farmacologia , Peixe-Zebra , Dedos de ZincoRESUMO
Neuronal-activity-regulated pentraxin (NARP/NPTX2/NP2) is a secreted synaptic protein that regulates the trafficking of glutamate receptors and mediates learning, memory, and drug addiction. The role of NPTX2 in regulating structural synaptic plasticity and behavior in a developing vertebrate is indefinite. We characterized the expression of nptx2a in larvae and adult zebrafish and established a transcription activator-like effector nuclease (TALEN)-mediated nptx2a mutant (nptx2a(-/-)) to study the role of Nptx2a in regulating structural synaptic plasticity and behavior. Similar to mammals, the zebrafish nptx2a was expressed in excitatory neurons in the brain and spinal cord. Its expression was induced in response to a mechanosensory stimulus but did not change during day and night. Behavioral assays showed that loss of Nptx2a results in reduced locomotor response to light-to-dark transition states and to a sound stimulus. Live imaging of synapses using the transgenic nptx2a:GAL4VP16 zebrafish and a fluorescent presynaptic synaptophysin (SYP) marker revealed reduced synaptic density in the axons of the spinal motor neurons and the anterodorsal lateral-line ganglion (gAD), which regulate locomotor activity and locomotor response to mechanosensory stimuli, respectively. These results suggest that Nptx2a affects locomotor response to external stimuli by mediating structural synaptic plasticity in excitatory neuronal circuits.
Assuntos
Proteína C-Reativa/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Plasticidade Neuronal/fisiologia , Proteínas de Peixe-Zebra/fisiologia , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/fisiologia , Estimulação Acústica , Animais , Animais Geneticamente Modificados , Proteína C-Reativa/deficiência , Proteína C-Reativa/genética , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Humanos , Larva/citologia , Larva/fisiologia , Mecanotransdução Celular , Atividade Motora , Neurônios Motores/fisiologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/genética , Estimulação Luminosa , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genéticaRESUMO
Growth arrest-specific 2-like 3 (Gas2l3) is a newly discovered cell cycle protein and a cytoskeleton orchestrator that binds both actin filament and microtubule networks. Studies of cultured mammalian cells established Gas2l3 as a regulator of the cell division process, in particular cytokinesis and cell abscission. Thus far, the role of Gas2l3 in vivo remains entirely unknown. In order to investigate Gas2l3 in developing vertebrates, we cloned the zebrafish gene. Spatiotemporal analysis of gas2l3 expression revealed a ubiquitous maternal transcript as well as a zygotic transcript primarily restricted to brain tissues. We next conducted a series of loss-of-function experiments, and searched for developmental anomalies at the end of the segmentation period. Our analysis revealed abnormal brain morphogenesis and ventricle formation in gas2l3 knockdown embryos. This signature phenotype could be rescued by elevated levels of gas2l3 RNA. At the tissue level, gas2l3 downregulation interferes with cell proliferation, suggesting that the cell cycle activities of Gas2l3 are essential for brain tissue homeostasis. Altogether, this study provides the first insight into the function of gas2l3 in vivo, demonstrating its essential role in brain development.
Assuntos
Encéfalo/embriologia , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Morfogênese/fisiologia , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/genética , Primers do DNA/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Microscopia de Fluorescência , Dados de Sequência Molecular , Morfogênese/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Especificidade da Espécie , Proteínas de Peixe-Zebra/genéticaRESUMO
Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor retardation characterized by neurological impairment and abnormal thyroid hormone (TH) levels. Mutations in the TH transporter, monocarboxylate transporter 8 (MCT8), are associated with AHDS. MCT8 knock-out mice exhibit impaired TH levels; however, they lack neurological defects. Here, the zebrafish mct8 gene and promoter were isolated, and mct8 promoter-driven transgenic lines were used to show that, similar to humans, mct8 is primarily expressed in the nervous and vascular systems. Morpholino-based knockdown and rescue experiments revealed that MCT8 is strictly required for neural development in the brain and spinal cord. This study shows that MCT8 is a crucial regulator during embryonic development and establishes the first vertebrate model for MCT8 deficiency that exhibits a neurological phenotype.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Deficiência Intelectual Ligada ao Cromossomo X/genética , Hipotonia Muscular/genética , Atrofia Muscular/genética , Mutação , Animais , Encéfalo/metabolismo , Modelos Animais de Doenças , Humanos , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Modelos Genéticos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Neurônios/patologia , Fenótipo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Medula Espinal/metabolismo , Simportadores , Glândula Tireoide/metabolismo , Hormônios Tireóideos/metabolismo , Peixe-ZebraRESUMO
Hypothyroidism is a common pathological condition characterized by insufficient activity of the thyroid hormones (THs), thyroxine (T4), and 3,5,3'-triiodothyronine (T3), in the whole body or in specific tissues. Hypothyroidism is associated with inadequate development of the intestine as well as gastrointestinal diseases. We used a zebrafish model of hypothyroidism to identify and characterize TH-modulated genes and cellular pathways controlling intestine development. In the intestine of hypothyroid juveniles and adults, the number of mucus-secreting goblet cells was reduced, and this phenotype could be rescued by T3 treatment. Transcriptome profiling revealed dozens of differentially expressed genes in the intestine of hypothyroid adults compared to controls. Notably, the expression of genes encoding to Fgf19 and its receptor Fgfr4 was markedly increased in the intestine of hypothyroid adults, and treatment with T3 normalized it. Blocking fibroblast growth factor (FGF) signaling, using an inducible dominant-negative Fgfr transgenic line, rescued the number of goblet cells in hypothyroid adults. These results show that THs inhibit the Fgf19-Fgfr4 signaling pathway, which is associated with inhibition of goblet cell differentiation in hypothyroidism. Both the TH and Fgf19-Fgfr4 signaling pathways can be pharmaceutical targets for the treatment of TH-related gastrointestinal diseases.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Células Caliciformes/metabolismo , Hipotireoidismo/metabolismo , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Tiroxina/metabolismo , Tri-Iodotironina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Proliferação de Células , Modelos Animais de Doenças , Fatores de Crescimento de Fibroblastos/genética , Células Caliciformes/citologia , Humanos , Hipotireoidismo/genética , Hipotireoidismo/fisiopatologia , Mucosa Intestinal/crescimento & desenvolvimento , Mucosa Intestinal/metabolismo , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais , Peixe-Zebra/genética , Peixe-Zebra/crescimento & desenvolvimento , Proteínas de Peixe-Zebra/genéticaRESUMO
Background: The thyroid hormones (THs) triiodothyronine (T3) and thyroxine (T4) are crucial regulators of brain development and function. Cell-specific transporter proteins facilitate TH uptake and efflux across the cell membrane, and insufficient TH transport causes hypothyroidism and mental retardation. Mutations in the TH transporters monocarboxylate transporter 8 (MCT8, SLC16A2) and the organic anion-transporting polypeptide 1C1 (OATP1C1, SLCO1C1) are associated with the psychomotor retardation Allan-Herndon-Dudley syndrome and juvenile neurodegeneration, respectively. Methods: To understand the mechanisms and test potential treatments for the recently discovered OATP1C1 deficiency, we established an oatp1c1 mutant (oatp1c1-/-) zebrafish. Results:oatp1c1 is expressed in endothelial cells, neurons, and astrocytes in zebrafish. The activity of the hypothalamic-pituitary-thyroid axis and behavioral locomotor activity increased in oatp1c1-/- larvae. Neuropathological analysis revealed structural alteration in radial glial cells and shorter neuronal axons in oatp1c1-/- larvae and adults. Notably, oatp1c1-/- and oatp1c1-/-Xmct8-/- adults exhibit an enlarged thyroid gland (goiter). Pharmacological assays showed that TH analogs, but not THs, can reduce the size and improve the color of the thyroid gland in adult mutant zebrafish. Conclusion: These results establish a vertebrate model for OATP1C1 deficiency that demonstrates endocrinological, neurological, and behavioral alterations mimicking findings observed in an OATP1C1-deficient patient. Further, the curative effect of TH analogs in the oatp1c1-/- zebrafish model may provide a lead toward a treatment modality in human patients.
Assuntos
Sistema Hipotálamo-Hipofisário/fisiologia , Mutação , Neurônios/fisiologia , Transportadores de Ânions Orgânicos/genética , Glândula Tireoide/fisiologia , Proteínas de Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Astrócitos/metabolismo , Comportamento Animal , Encéfalo/metabolismo , Membrana Celular/metabolismo , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Técnicas de Inativação de Genes , Microscopia de Fluorescência , Transportadores de Ânions Orgânicos/deficiência , Transportadores de Ânions Orgânicos/fisiologia , Peixe-Zebra , Proteínas de Peixe-Zebra/fisiologiaRESUMO
Sleep is tightly regulated by the circadian clock and homeostatic mechanisms. Although the sleep/wake cycle is known to be associated with structural and physiological synaptic changes that benefit the brain, the function of sleep is still debated. The hypothalamic hypocretin/orexin (Hcrt) neurons regulate various functions including feeding, reward, sleep, and wake. Continuous imaging of single neuronal circuits in live animals is vital to understanding the role of sleep in regulating synaptic dynamics, and the transparency of the zebrafish model enables time-lapse imaging of single synapses during both day and night. Here, we use the gephyrin (Gphnb) protein, a central inhibitory synapse organizer, as a fluorescent post-synaptic marker of inhibitory synapses. Double labeling showed that Gphnb-tagRFP and collybistin-EGFP clusters co-localized in dendritic inhibitory synapses. Using a transgenic hcrt:Gphnb-EGFP zebrafish, we showed that the number of inhibitory synapses in the dendrites of Hcrt neurons was increased during development. To determine the effect of sleep on the inhibitory synapses, we performed two-photon live imaging of Gphnb-EGFP in Hcrt neurons during day and night, under light/dark and constant light and dark conditions, and following sleep deprivation (SD). We found that synapse number increased during the night under light/dark conditions but that these changes were eliminated under constant light or dark conditions. SD reduced synapse number during the night, and the number increased during post-deprivation daytime sleep rebound. These results suggest that rhythmic structural plasticity of inhibitory synapses in Hcrt dendrites is independent of the circadian clock and is modulated by consolidated wake and sleep.
Assuntos
Dendritos/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Sono/fisiologia , Sinapses/fisiologia , Animais , Animais Geneticamente Modificados , Relógios Circadianos/fisiologia , Hipotálamo/fisiologia , Inibição Neural/fisiologia , Orexinas/metabolismo , Peixe-ZebraRESUMO
Hypomyelination is a key symptom of Allan-Herndon-Dudley syndrome (AHDS), a psychomotor retardation associated with mutations in the thyroid-hormone (TH) transporter MCT8 (monocarboxylate transporter 8). AHDS is characterized by severe intellectual deficiency, neuromuscular impairment and brain hypothyroidism. In order to understand the mechanism for TH-dependent hypomyelination, we developed an mct8 mutant (mct8-/-) zebrafish model. The quantification of genetic markers for oligodendrocyte progenitor cells (OPCs) and mature oligodendrocytes revealed reduced differentiation of OPCs into oligodendrocytes in mct8-/- larvae and adults. Live imaging of single glial cells showed that the number of oligodendrocytes and the length of their extensions are reduced, and the number of peripheral Schwann cells is increased, in mct8-/- larvae compared with wild type. Pharmacological analysis showed that TH analogs and clemastine partially rescued the hypomyelination in the CNS of mct8-/- larvae. Intriguingly, triiodothyronine (T3) treatment rescued hypomyelination in mct8-/- embryos before the maturation of the blood-brain barrier (BBB), but did not affect hypomyelination in older larvae. Thus, we expressed Mct8-tagRFP in the endothelial cells of the vascular system and showed that even relatively weak mosaic expression completely rescued hypomyelination in mct8-/- larvae. These results suggest potential pharmacological treatments and BBB-targeted gene therapy that can enhance myelination in AHDS and possibly in other TH-dependent brain disorders.
Assuntos
Barreira Hematoencefálica/patologia , Clemastina/farmacologia , Terapia Genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Peixe-Zebra/genética , Animais , Biomarcadores/metabolismo , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Larva/efeitos dos fármacos , Larva/genética , Transportadores de Ácidos Monocarboxílicos/deficiência , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Células de Schwann/efeitos dos fármacos , Células de Schwann/metabolismo , Células de Schwann/patologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Hormônios Tireóideos/agonistas , Hormônios Tireóideos/metabolismoRESUMO
Neurotensin (NTS) is a 13 amino acid neuropeptide that is expressed in the hypothalamus. In mammals, NTS-producing neurons that express leptin receptor (LepRb) regulate the function of hypocretin/orexin (HCRT) and dopamine neurons. Thus, the hypothalamic leptin-NTS-HCRT neuronal network orchestrates key homeostatic output, including sleep, feeding, and reward. However, the intricate mechanisms of the circuitry and the unique role of NTS-expressing neurons remain unclear. We studied the NTS neuronal networks in zebrafish and cloned the genes encoding the NTS neuropeptide and receptor (NTSR). Similar to mammals, the ligand is expressed primarily in the hypothalamus, while the receptor is expressed widely throughout the brain in zebrafish. A portion of hypothalamic nts-expressing neurons are inhibitory and some coexpress leptin receptor (lepR1). As in mammals, NTS and HCRT neurons are localized adjacently in the hypothalamus. To track the development and axonal projection of NTS neurons, the NTS promoter was isolated. Transgenesis and double labeling of NTS and HCRT neurons showed that NTS axons project toward HCRT neurons, some of which express ntsr. Moreover, another target of NTS neurons is ntsr-expressing dopaminergeric neurons. These findings suggest structural circuitry between leptin, NTS, and hypocretinergic or dopaminergic neurons and establish the zebrafish as a model to study the role of these neuronal circuits in the regulation of feeding, sleep, and reward.
Assuntos
Hipotálamo/anatomia & histologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Leptina/metabolismo , Neuropeptídeos/metabolismo , Neurotensina/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/anatomia & histologia , Animais , Animais Geneticamente Modificados , Dopamina/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipotálamo/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Vias Neurais/anatomia & histologia , Vias Neurais/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Orexinas , Receptores para Leptina/metabolismo , Receptores de Neurotensina/metabolismo , Peixe-Zebra/metabolismoRESUMO
Sleep has been conserved throughout evolution; however, the molecular and neuronal mechanisms of sleep are largely unknown. The hypothalamic hypocretin/orexin (Hcrt) neurons regulate sleep\wake states, feeding, stress, and reward. To elucidate the mechanism that enables these various functions and to identify sleep regulators, we combined fluorescence cell sorting and RNA-seq in hcrt:EGFP zebrafish. Dozens of Hcrt-neuron-specific transcripts were identified and comprehensive high-resolution imaging revealed gene-specific localization in all or subsets of Hcrt neurons. Clusters of Hcrt-neuron-specific genes are predicted to be regulated by shared transcription factors. These findings show that Hcrt neurons are heterogeneous and that integrative molecular mechanisms orchestrate their diverse functions. The voltage-gated potassium channel Kcnh4a, which is expressed in all Hcrt neurons, was silenced by the CRISPR-mediated gene inactivation system. The mutant kcnh4a (kcnh4a(-/-)) larvae showed reduced sleep time and consolidation, specifically during the night, suggesting that Kcnh4a regulates sleep.