RESUMO
Neuroendocrine prostate cancer is a rapidly progressive and lethal disease characterized by early visceral metastasis, poor prognosis, and limited treatment options. Uncovering the oncogenic mechanisms could lead to the discovery of potential therapeutic avenues. Here, we demonstrate that the RNA-binding protein ELAVL3 is specifically upregulated in neuroendocrine prostate cancer and that overexpression of ELAVL3 alone is sufficient to induce the neuroendocrine phenotype in prostate adenocarcinoma. Mechanistically, ELAVL3 is transcriptionally regulated by MYCN and subsequently binds to and stabilizes MYCN and RICTOR mRNA. Moreover, ELAVL3 is shown to be released in extracellular vesicles and induce neuroendocrine differentiation of adenocarcinoma cells via an intercellular mechanism. Pharmacological inhibition of ELAVL3 with pyrvinium pamoate, an FDA-approved drug, effectively suppresses tumor growth, reduces metastatic risk, and improves survival in neuroendocrine prostate cancer mouse models. Our results identify ELAVL3 as a critical regulator of neuroendocrine differentiation in prostate cancer and propose a drug repurposing strategy for targeted therapies.
Assuntos
Adenocarcinoma , Neoplasias da Próstata , Humanos , Masculino , Animais , Camundongos , Proteína Proto-Oncogênica N-Myc/genética , Retroalimentação , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Fenótipo , Adenocarcinoma/genética , Linhagem Celular Tumoral , Proteína Semelhante a ELAV 3/genéticaRESUMO
Amyotrophic lateral sclerosis is a progressive fatal neurodegenerative disease caused by loss of motor neurons and characterized neuropathologically in almost all cases by nuclear depletion and cytoplasmic aggregation of TDP-43, a nuclear RNA-binding protein (RBP). We identified ELAVL3 as one of the most downregulated genes in our transcriptome profiles of laser captured microdissection of motor neurons from sporadic ALS nervous systems and the most dysregulated of all RBPs. Neuropathological characterizations showed ELAVL3 nuclear depletion in a great percentage of remnant motor neurons, sometimes accompanied by cytoplasmic accumulations. These abnormalities were common in sporadic cases with and without intermediate expansions in ATXN2 and familial cases carrying mutations in C9orf72 and SOD1. Depletion of ELAVL3 occurred at both the RNA and protein levels and a short protein isoform was identified, but it is not related to a TDP-43-dependent cryptic exon in intron 3. Strikingly, ELAVL3 abnormalities were more frequent than TDP-43 abnormalities and occurred in motor neurons still with normal nuclear TDP-43 present, but all neurons with abnormal TDP-43 also had abnormal ELAVL3. In a neuron-like cell culture model using SH-SY5Y cells, ELAVL3 mislocalization occurred weeks before TDP-43 abnormalities were seen. We interrogated genetic databases, but did not identify association of ELAVL3 genetic structure with ALS. Taken together, these findings suggest that ELAVL3 is an important RBP in ALS pathogenesis acquired early and the neuropathological data suggest that it is involved by loss of function rather than cytoplasmic toxicity.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Proteína Semelhante a ELAV 3/metabolismo , Neurônios Motores/metabolismo , Núcleo Celular/metabolismo , HumanosRESUMO
Deregulated full-length anaplastic lymphoma kinase (ALK) overexpression has been found in some primary solid tumors, but little is known about its role in ovarian high-grade serous carcinoma (HGSC). The current study focused on the functional roles of ALK in HGSC. Cytoplasmic ALK immunoreactivity without chromosomal rearrangement and gene mutations was significantly higher in HGSC compared with non-HGSC-type ovarian carcinomas, and was significantly associated with several unfavorable clinicopathologic factors and poor prognosis. HGSC cell lines stably overexpressing ALK exhibited increased cell proliferation, enhanced cancer stem cell features, and accelerated cell mobility, whereas these phenotypes were abrogated in ALK-knockdown cells. Expression of the nervous system-associated gene, ELAVL3, and the corresponding protein (commonly known as HuC) was significantly increased in cells overexpressing ALK. Expression of SRY-box transcription factor (Sox)2 and Sox3 (genes associated with the neural progenitor population) increased in ALK-overexpressing but not ALK-knockdown cells. Furthermore, overexpression of Sox2 or Sox3 enhanced both ALK and ELAVL3 promoter activities, suggesting the existence of ALK/Sox/HuC signaling loops. Finally, ALK overexpression was attributed to increased expression of neuroendocrine markers, including synaptophysin, CD56, and B-cell lymphoma 2, in HGSC tissues. These findings suggest that overexpression of full-length ALK may influence the biological behavior of HGSC through cooperation with ELAVL3 and Sox factors, leading to the establishment and maintenance of the aggressive phenotypic characteristics of HGSC.
Assuntos
Quinase do Linfoma Anaplásico/metabolismo , Cistadenocarcinoma Seroso/enzimologia , Cistadenocarcinoma Seroso/patologia , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Adulto , Idoso , Diferenciação Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Citoplasma/enzimologia , Proteína Semelhante a ELAV 3/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , Modelos Biológicos , Análise Multivariada , Gradação de Tumores , Células-Tronco Neoplásicas/patologia , Células Neuroendócrinas/metabolismo , Células Neuroendócrinas/patologia , Fenótipo , Prognóstico , Intervalo Livre de Progressão , Fatores de Transcrição SOX/metabolismoRESUMO
Neuron-enriched microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124), direct cell fate switching of human fibroblasts to neurons when ectopically expressed by repressing antineurogenic genes. How these miRNAs function after the repression of fibroblast genes for neuronal fate remains unclear. Here, we identified targets of miR-9/9*-124 as reprogramming cells activate the neuronal program and reveal the role of miR-124 that directly promotes the expression of its target genes associated with neuronal development and function. The mode of miR-124 as a positive regulator is determined by the binding of both AGO and a neuron-enriched RNA-binding protein, ELAVL3, to target transcripts. Although existing literature indicates that miRNA-ELAVL family protein interaction can result in either target gene up-regulation or down-regulation in a context-dependent manner, we specifically identified neuronal ELAVL3 as the driver for miR-124 target gene up-regulation in neurons. In primary human neurons, repressing miR-124 and ELAVL3 led to the down-regulation of genes involved in neuronal function and process outgrowth and cellular phenotypes of reduced inward currents and neurite outgrowth. Our results highlight the synergistic role between miR-124 and RNA-binding proteins to promote target gene regulation and neuronal function.
Assuntos
Proteína Semelhante a ELAV 3/biossíntese , Regulação da Expressão Gênica , MicroRNAs/metabolismo , Neurônios/metabolismo , Adulto , Proteína Semelhante a ELAV 3/genética , Feminino , Humanos , MicroRNAs/genéticaRESUMO
Development of the retina is regulated by growth factors, such as insulin-like growth factors 1 and 2 (IGF-1/2), which coordinate proliferation, differentiation, and maturation of the neuroepithelial precursors cells. In the circulation, IGF-1/2 are transported by the insulin growth factor binding proteins (IGFBPs) family members. IGFBPs can impact positively and negatively on IGF-1, by making it available or sequestering IGF-1 to or from its receptor. In this study, we investigated the expression of IGFBPs and their role in the generation of human retinal organoids from human pluripotent stem cells, showing a dynamic expression pattern suggestive of different IGFBPs being used in a stage-specific manner to mediate IGF-1 functions. Our data show that IGF-1 addition to culture media facilitated the generation of retinal organoids displaying the typical laminated structure and photoreceptor maturation. The organoids cultured in the absence of IGF-1, lacked the typical laminated structure at the early stages of differentiation and contained significantly less photoreceptors and more retinal ganglion cells at the later stages of differentiation, confirming the positive effects of IGF-1 on retinal lamination and photoreceptor development. The organoids cultured with the IGFBP inhibitor (NBI-31772) and IGF-1 showed lack of retinal lamination at the early stages of differentiation, an increased propensity to generate horizontal cells at mid-stages of differentiation and reduced photoreceptor development at the later stages of differentiation. Together these data suggest that IGFBPs enable IGF-1's role in retinal lamination and photoreceptor development in a stage-specific manner.
Assuntos
Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Fator de Crescimento Insulin-Like II/genética , Fator de Crescimento Insulin-Like I/genética , Organoides/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Células-Tronco Pluripotentes/metabolismo , Catecóis/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proteína Semelhante a ELAV 3/genética , Proteína Semelhante a ELAV 3/metabolismo , Proteína Semelhante a ELAV 4/genética , Proteína Semelhante a ELAV 4/metabolismo , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/antagonistas & inibidores , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like II/metabolismo , Isoquinolinas/farmacologia , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Organoides/citologia , Organoides/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/citologia , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Recoverina/genética , Recoverina/metabolismo , Transdução de Sinais , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , gama-Sinucleína/genética , gama-Sinucleína/metabolismoRESUMO
Noise is prevalent in biology and has been widely quantified using snapshot measurements. This static view obscures our understanding of dynamic noise properties and how these affect gene expression and cell state transitions. Using a CRISPR/Cas9 Zebrafish her6::Venus reporter combined with mathematical and in vivo experimentation, we explore how noise affects the protein dynamics of Her6, a basic helix-loop-helix transcriptional repressor. During neurogenesis, Her6 expression transitions from fluctuating to oscillatory at single-cell level. We identify that absence of miR-9 input generates high-frequency noise in Her6 traces, inhibits the transition to oscillatory protein expression and prevents the downregulation of Her6. Together, these impair the upregulation of downstream targets and cells accumulate in a normally transitory state where progenitor and early differentiation markers are co-expressed. Computational modelling and double smFISH of her6 and the early neurogenesis marker, elavl3, suggest that the change in Her6 dynamics precedes the downregulation in Her6 levels. This sheds light onto the order of events at the moment of cell state transition and how this is influenced by the dynamic properties of noise. Our results suggest that Her/Hes oscillations, facilitated by dynamic noise optimization by miR-9, endow progenitor cells with the ability to make a cell state transition.
Assuntos
Animais Geneticamente Modificados/embriologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Relógios Biológicos , MicroRNAs/metabolismo , Neurogênese , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Animais Geneticamente Modificados/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteína Semelhante a ELAV 3/genética , Proteína Semelhante a ELAV 3/metabolismo , MicroRNAs/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The neuron-specific Elav-like Hu RNA-binding proteins were described to play an important role in neuronal differentiation and plasticity by ensuring the post-transcriptional control of RNAs encoding for various proteins. Although Elav-like Hu proteins alterations were reported in diabetes or neuropathy, little is known about the regulation of neuron-specific Elav-like Hu RNA-binding proteins in sensory neurons of dorsal root ganglia (DRG) due to the diabetic condition. The goal of our study was to analyze the gene and protein expression of HuB, HuC, and HuD in DRG sensory neurons in diabetes. The diabetic condition was induced in CD-1 adult male mice with single-intraperitoneal injection of streptozotocin (STZ, 150 mg/kg), and 8-weeks (advanced diabetes) after induction was quantified the Elav-like proteins expression. Based on the glycemia values, we identified two types of responses to STZ, and mice were classified in STZ-resistant (diabetic resistant, glycemia < 260 mg/dL) and STZ-sensitive (diabetic, glycemia > 260 mg/dL). Body weight measurements indicated that 8-weeks after STZ-induction of diabetes, control mice have a higher increase in body weight compared to the diabetic and diabetic resistant mice. Moreover, after 8-weeks, diabetic mice (19.52 ± 3.52 s) have longer paw withdrawal latencies in the hot-plate test than diabetic resistant (11.36 ± 1.92 s) and control (11.03 ± 1.97 s) mice, that correlates with the installation of warm hypoalgesia due to the diabetic condition. Further on, we evidenced the decrease of Elav-like gene expression in DRG neurons of diabetic mice (Elavl2, 0.68 ± 0.05 fold; Elavl3, 0.65 ± 0.01 fold; Elavl4, 0.53 ± 0.07 fold) and diabetic resistant mice (Ealvl2, 0.56 ± 0.07 fold; Elavl3, 0.32 ± 0.09 fold) compared to control mice. Interestingly, Elav-like genes have a more accentuated downregulation in diabetic resistant than in diabetic mice, although hypoalgesia was evidenced only in diabetic mice. The Elav-like gene expression changes do not always correlate with the Hu protein expression changes. To detail, HuB is upregulated and HuD is downregulated in diabetic mice, while HuB, HuC, and HuD are downregulated in diabetic resistant mice compared to control mice. To resume, we demonstrated HuD downregulation and HuB upregulation in DRG sensory neurons induced by diabetes, which might be correlated with altered post-transcriptional control of RNAs involved in the regulation of thermal hypoalgesia condition caused by the advanced diabetic neuropathy.
Assuntos
Proteína Semelhante a ELAV 2/genética , Proteína Semelhante a ELAV 3/genética , Proteína Semelhante a ELAV 4/genética , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Células Receptoras Sensoriais/metabolismo , Animais , Biomarcadores , Glicemia , Peso Corporal , Diabetes Mellitus Experimental , Proteína Semelhante a ELAV 2/metabolismo , Proteína Semelhante a ELAV 3/metabolismo , Proteína Semelhante a ELAV 4/metabolismo , Gânglios Espinais/fisiopatologia , Imuno-Histoquímica , Camundongos , Proteínas de Ligação a RNARESUMO
The RNA-binding protein HuC/D displays a neuron-specific expression and is involved in neuronal differentiation and the maintenance of the nervous system. Here we investigated the diagnostic value of HuC/D in neuroblastomas. We evaluated 85 neuroblastic tumors: 81 neuroblastomas; 3 ganglioneuroblastomas, intermixed; 1 ganglioneuroma, maturing; and 101 other tumors consisting of 34 Ewing sarcomas, 14 nephroblastomas, 11 rhabdomyosarcomas, 15 pulmonary small cell carcinomas, 18 pancreatic neuroendocrine tumors, and 9 pheochromocytomas. Immunohistochemistry for HuC/D, PHOX2B, and tyrosine hydroxylase was performed. The immunoreactivity for HuC/D was semiquantified using the total score (TS; range, 0-8). HuC/D positivity was defined as a TS ≥6. The TS of the neuroblastic tumors (mean TS, 7.94) was significantly higher than those of the other small round cell tumors and neuroendocrine tumors (P < .001) except for the pheochromocytomas (mean TS, 6.89; P = .074). HuC/D was positive in all 85 neuroblastic tumors, 1 (2.9%) Ewing sarcoma, 1 (6.7%) pulmonary small cell carcinoma, and 8 (89%) pheochromocytomas. PHOX2B was positive in all of the neuroblastic tumors and pheochromocytomas. Tyrosine hydroxylase was positive in 80 (94%) neuroblastic tumors, 1 (9.1%) rhabdomyosarcoma, and all of the pheochromocytomas. Therefore, HuC/D serves as a highly sensitive diagnostic marker to distinguish neuroblastomas from other small round cell tumors. The combination of HuC/D and PHOX2B staining may be valuable for the diagnosis of neuroblastic tumors, especially in the assessment of small sections. HuC/D expression in tumors may be related to catecholamine production or a neural crest-derived cell origin.
Assuntos
Neoplasias das Glândulas Suprarrenais/diagnóstico , Carcinoma de Células Pequenas/diagnóstico , Proteína Semelhante a ELAV 3/metabolismo , Ganglioneuroblastoma/diagnóstico , Neuroblastoma/diagnóstico , Tumores Neuroendócrinos/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Feocromocitoma/diagnóstico , Adolescente , Neoplasias das Glândulas Suprarrenais/metabolismo , Neoplasias das Glândulas Suprarrenais/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/patologia , Criança , Pré-Escolar , Diagnóstico Diferencial , Feminino , Ganglioneuroblastoma/metabolismo , Ganglioneuroblastoma/patologia , Proteínas de Homeodomínio/metabolismo , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Feocromocitoma/metabolismo , Feocromocitoma/patologia , Sensibilidade e Especificidade , Fatores de Transcrição/metabolismo , Adulto JovemRESUMO
Voltage-sensitive dye (VSD) imaging enables fast, direct, and simultaneous detection of membrane potentials from a population of neurons forming neuronal circuits. This enables the detection of hyperpolarization together with depolarization, whose balance plays a pivotal role in the function of many brain regions. Among these is the cerebellum, which contains a significant number of inhibitory neurons. However, the mechanism underlying the functional development remains unclear. In this study, we used a model system ideal to study neurogenesis by applying VSD imaging to the cerebellum of zebrafish larvae to analyze the neuronal activity of the developing cerebellum, focusing on both excitation and inhibition. We performed in-vivo high-speed imaging of the entire cerebellum of the zebrafish, which was stained using Di-4-ANEPPS, a widely used VSD. To examine whether neuronal activity in the zebrafish cerebellum could be detected by this VSD, we applied electrical stimulation during VSD imaging, which showed that depolarization was detected widely in the cerebellum upon stimulation. These responses mostly disappeared following treatment with tetrodotoxin, indicating that Di-4-ANEPPS enabled optical measurement of neuronal activity in the developing cerebellum of zebrafish. Moreover, hyperpolarizing signals were also detected upon stimulation, but these were significantly reduced by treatment with picrotoxin, a GABAA receptor inhibitor, indicating that these responses represent inhibitory signals. This approach will enable a detailed analysis of the spatiotemporal dynamics of the excitation and inhibition in the cerebellum along its developmental stages, leading to a deeper understanding of the functional development of the cerebellum in vertebrates.
Assuntos
Cerebelo/citologia , Cerebelo/crescimento & desenvolvimento , Neurônios/fisiologia , Imagens com Corantes Sensíveis à Voltagem/métodos , Animais , Animais Geneticamente Modificados , Proteína Semelhante a ELAV 3/genética , Proteína Semelhante a ELAV 3/metabolismo , Estimulação Elétrica , Antagonistas GABAérgicos/farmacologia , Larva , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Neurônios/efeitos dos fármacos , Picrotoxina/farmacologia , Compostos de Piridínio/metabolismo , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Peixe-Zebra , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Currently available antidepressant drugs often fail to achieve full remission and patients might evolve to treatment resistance, showing the need to achieve a better therapy of depressive disorders. Increasing evidence supports that post-transcriptional regulation of gene expression is important in neuronal development and survival and a relevant role is played by RNA binding proteins (RBP). To explore new therapeutic strategies, we investigated the role of the neuron-specific ELAV-like RBP (HuB, HuC, HuD) in a mouse model of depression. In this study, a 4-week unpredictable chronic mild stress (UCMS) protocol was applied to mice to induce a depressive-like phenotype. In the last 2 weeks of the UCMS regimen, silencing of HuB, HuC or HuD was performed by using specific antisense oligonucleotides (aODN). Treatment of UCMS-exposed mice with anti-HuB and anti-HuC aODN improved both anhedonia and behavioural despair, used as measures of depressive-like behaviour, without modifying the response of stressed mice to an anxiety-inducing environment. On the contrary, HuD silencing promoted an anxiolytic-like behaviour in UCMS-exposed mice without improving depressive-like behaviours. The antidepressant-like phenotype of anti-HuB and anti-HuC mice was not shown concurrently with the promotion of adult hippocampal neurogenesis in the dentate gyrus, and no increase in the BDNF and CREB content was detected. Conversely, in the CA3 hippocampal region, projection area of newly born neurons, HuB and HuC silencing increased the number of BrdU/NeuN positive cells. These results give the first indication of a role of nELAV in the modulation of emotional states in a mouse model of depression.
Assuntos
Antidepressivos/farmacologia , Transtorno Depressivo/tratamento farmacológico , Proteína Semelhante a ELAV 2/antagonistas & inibidores , Proteína Semelhante a ELAV 3/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Anedonia/efeitos dos fármacos , Anedonia/fisiologia , Animais , Ansiedade/metabolismo , Bromodesoxiuridina , Proteínas de Ligação a DNA , Transtorno Depressivo/metabolismo , Transtorno Depressivo/patologia , Modelos Animais de Doenças , Proteína Semelhante a ELAV 2/metabolismo , Proteína Semelhante a ELAV 3/metabolismo , Proteína Semelhante a ELAV 4/antagonistas & inibidores , Proteína Semelhante a ELAV 4/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/efeitos dos fármacos , Neurogênese/fisiologia , Neurônios/metabolismo , Neurônios/patologia , Proteínas Nucleares/metabolismo , Distribuição AleatóriaRESUMO
Alternative splicing of RNAs diversifies the functionalities of proteins, and it is optimized for each cell type and each developmental stage. nElavl (composed of Elavl2, Elavl3, and Elavl4) proteins are the RNA-binding proteins that is specifically expressed in neurons, regulate the alternative splicing of target RNAs, and promote neuronal differentiation and maturation. Recent studies revealed that Elavl3 knockout (Elavl3-/-) mice completely lost the expression of nElavl proteins in the Purkinje cells and exhibited cerebellar dysfunction. Here, we found that the alternative splicing of AnkyrinG exon 34 was misregulated in the cerebella of Elavl3-/- mice. AnkyrinG is an essential factor for the formation of neuronal polarity and is required for normal neuronal functions. We revealed that exon 34 of AnkyrinG was normally included in immature neurons and was mostly excluded in mature neurons; however, it was included in the cerebella of Elavl3-/- mice even in adulthood. In the Purkinje cells of adult Elavl3-/- mice, the length of the AnkyrinG-positive region shortened and somatic organelles leaked into the axons. These results suggested that exon 34 of AnkyrinG is an embryonic-stage-preferential exon that should be excluded from mature neurons and that Elavl3 regulates neuronal polarity through alternative splicing of this exon.
Assuntos
Anquirinas/genética , Proteína Semelhante a ELAV 3/genética , Éxons , Células de Purkinje/fisiologia , Processamento Alternativo , Sequência de Aminoácidos , Animais , Anquirinas/metabolismo , Polaridade Celular/genética , Doenças Cerebelares/genética , Doenças Cerebelares/metabolismo , Doenças Cerebelares/patologia , Cerebelo/patologia , Proteína Semelhante a ELAV 3/metabolismo , Proteína Semelhante a ELAV 3/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células de Purkinje/citologia , Células de Purkinje/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Gastrointestinal symptoms are the first signs of fluoride (F) toxicity. In the present study, the jejunum of rats chronically exposed to F was evaluated by proteomics, as well as by morphological analysis. Wistar rats received water containing 0, 10 or 50 mgF/L during 30 days. HuC/D, neuronal Nitric Oxide (nNOS), Vasoactive Intestinal Peptide (VIP), Calcitonin Gene Related Peptide (CGRP), and Substance P (SP) were detected in the myenteric plexus of the jejunum by immunofluorescence. The density of nNOS-IR neurons was significantly decreased (compared to both control and 10 mgF/L groups), while the VIP-IR varicosities were significantly increased (compared to control) in the group treated with the highest F concentration. Significant morphological changes were seen observed in the density of HUC/D-IR neurons and in the area of SP-IR varicosities for F-treated groups compared to control. Changes in the abundance of various proteins correlated with relevant biological processes, such as protein synthesis, glucose homeostasis and energy metabolism were revealed by proteomics.
Assuntos
Fluoretos/efeitos adversos , Jejuno/efeitos dos fármacos , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Duodeno/metabolismo , Proteína Semelhante a ELAV 3/metabolismo , Sistema Nervoso Entérico/efeitos dos fármacos , Intestino Delgado/metabolismo , Masculino , Minerais/metabolismo , Óxido Nítrico/análise , Óxido Nítrico/metabolismo , Proteômica/métodos , Ratos , Ratos Wistar , Substância P/metabolismo , Peptídeo Intestinal Vasoativo/metabolismoRESUMO
AIMS: Serotonergic (5-HT) modulation of the lateral habenula (LHb) activity is central in normal and pathologic conditions such as mood disorders. Among the multiple 5-HT receptors (5-HTRs) involved, the 5-HT2C R seems to play a pivotal role. Yet, the role of 5-HT2A Rs in the control of the LHb neuronal activity is completely unknown. METHODS: Single-cell extracellular recording of the LHb neurons was used in rats to study the effect of the general activation and blockade of the 5-HT2C R and 5-HT2A R with Ro 60-0175 and SB242084, TCB-2 and MDL11939, respectively. The expression of both receptors in the LHb was confirmed using immunohistochemistry. RESULTS: Cumulative doses (5-640 µg/kg, iv) of Ro 60-0175 and TCB-2 affected the activity of 34% and 63% of the LHb recorded neurons, respectively. LHb neurons were either inhibited at low doses or excited at higher doses of the 5-HT2A/C R agonists. SB242084 or MDL11939 (both at 200 µg/kg, iv) did not modify neuronal firing when injected alone, but reverted the bidirectional effects of Ro 60-0175 or TCB-2, respectively. 5-HT2C Rs and 5-HT2A Rs are expressed in less than the 20% of the LHb neurons, and they neither colocalize nor make heterodimers. Strikingly, only 5-HT2A Rs are expressed by the majority of LHb astrocyte cells. CONCLUSIONS: Peripheral administration of 5-HT2A R agonist promotes a heterogeneous pattern of neuronal responses in the LHb, and these effects are more prominent than those induced by the 5-HT2C R activation.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Habenula/citologia , Habenula/metabolismo , Neurônios/fisiologia , Receptor 5-HT2A de Serotonina/metabolismo , Receptor 5-HT2C de Serotonina/metabolismo , Animais , Relação Dose-Resposta a Droga , Proteína Semelhante a ELAV 3/metabolismo , Glutamato Descarboxilase/metabolismo , Habenula/efeitos dos fármacos , Masculino , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Serotoninérgicos/farmacologiaRESUMO
The use of electronic cigarettes (e-cigarettes) is increasing despite insufficient information concerning their long-term effects, including the effects of maternal e-cigarette use on pre- and postnatal development. Our previous study demonstrated that developmental exposure to 1,2-propanediol (a principal component of e-cigarette liquid) affected early development of zebrafish, causing reduced growth, deformities, and hyperactive swimming behavior in larvae. The current study extends assessment of the developmental toxicity of 1,2-propanediol by examining additional long-term behavioral effects. We demonstrate that embryonic/larval exposure of zebrafish to 1,2-propanediol (0.625% or 1.25%) not only affected behavioral parameters in the larvae, but also caused persisting behavioral effects in adults after early developmental exposure. Additional parameters, including neural and vascular development in larvae, stress response in adults, and concentration of neurotransmitters dopamine and serotonin in adult brain were examined, in order to explain the behavioral differences. These additional assessments did not find 1,2-propanediol exposure to significantly affect Tg(Neurog1:GFP) or the transcript abundance of neural genes (Neurog1, Ascl1a, Elavl3, and Lef1). Vascular development was not found to be affected by 1,2-propanediol exposure, as inferred from experiments with Tg(Flk1:eGFP) zebrafish; however, transcript abundance of vascular genes (Flk1, Vegf, Tie-2, and Angpt1) was decreased. No statistically significant changes were noted for plasma cortisol or brain neurotransmitters in adult fish. Lastly, analysis of gene transcripts involved with 1,2-propanediol metabolism (Adh5, Aldh2.1, and Ldha) showed an increase in Adh5 transcript. This is the first study to demonstrate that developmental exposure to 1,2-propanediol has long-term neurobehavioral consequences in adult zebrafish, showing that e-cigarettes contain substances potentially harmful to neurodevelopment.
Assuntos
Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Propilenoglicol/toxicidade , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/metabolismo , Dopamina/metabolismo , Proteína Semelhante a ELAV 3/metabolismo , Hidrocortisona/sangue , Inativação Metabólica/genética , Proteínas do Tecido Nervoso/metabolismo , Serotonina/metabolismo , Fatores de Transcrição/metabolismo , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
Neuronal Elav-like (nElavl or neuronal Hu) proteins are RNA-binding proteins that regulate RNA stability and alternative splicing, which are associated with axonal and synaptic structures. nElavl proteins promote the differentiation and maturation of neurons via their regulation of RNA. The functions of nElavl in mature neurons are not fully understood, although Elavl3 is highly expressed in the adult brain. Furthermore, possible associations between nElavl genes and several neurodegenerative diseases have been reported. We investigated the relationship between nElavl functions and neuronal degeneration using Elavl3-/- mice. Elavl3-/- mice exhibited slowly progressive motor deficits leading to severe cerebellar ataxia, and axons of Elavl3-/- Purkinje cells were swollen (spheroid formation), followed by the disruption of synaptic formation of axonal terminals. Deficit in axonal transport and abnormalities in neuronal polarity was observed in Elavl3-/- Purkinje cells. These results suggest that nElavl proteins are crucial for the maintenance of axonal homeostasis in mature neurons. Moreover, Elavl3-/- mice are unique animal models that constantly develop slowly progressive axonal degeneration. Therefore, studies of Elavl3-/- mice will provide new insight regarding axonal degenerative processes.
Assuntos
Axônios/patologia , Ataxia Cerebelar/etiologia , Proteína Semelhante a ELAV 3/fisiologia , Transtornos Motores/etiologia , Degeneração Neural/etiologia , Neurônios/patologia , Células de Purkinje/patologia , Animais , Transporte Axonal , Axônios/metabolismo , Células Cultivadas , Ataxia Cerebelar/metabolismo , Ataxia Cerebelar/patologia , Regulação da Expressão Gênica , Cinesinas/genética , Cinesinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Transtornos Motores/metabolismo , Transtornos Motores/patologia , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurônios/metabolismo , Células de Purkinje/metabolismoRESUMO
In the developing hypothalamus, the fat-derived hormone leptin stimulates the growth of axons from the arcuate nucleus of the hypothalamus (ARH) to other regions that control energy balance. These projections are significantly reduced in leptin deficient (Lepob/ob ) mice and this phenotype is largely rescued by neonatal leptin treatments. However, treatment of mature Lepob/ob mice is ineffective, suggesting that the trophic action of leptin is limited to a developmental critical period. To temporally delineate closure of this critical period for leptin-stimulated growth, we treated Lepob/ob mice with exogenous leptin during a variety of discrete time periods, and measured the density of Agouti-Related Peptide (AgRP) containing projections from the ARH to the ventral part of the dorsomedial nucleus of the hypothalamus (DMHv), and to the medial parvocellular part of the paraventricular nucleus (PVHmp). The results indicate that leptin loses its neurotrophic potential at or near postnatal day 28. The duration of leptin exposure appears to be important, with 9- or 11-day treatments found to be more effective than shorter (5-day) treatments. Furthermore, leptin treatment for 9 days or more was sufficient to restore AgRP innervation to both the PVHmp and DMHv in Lepob/ob females, but only to the DMHv in Lepob/ob males. Together, these findings reveal that the trophic actions of leptin are contingent upon timing and duration of leptin exposure, display both target and sex specificity, and that modulation of leptin-dependent circuit formation by each of these factors may carry enduring consequences for feeding behavior, metabolism, and obesity risk.
Assuntos
Proteína Relacionada com Agouti/metabolismo , Núcleo Arqueado do Hipotálamo/citologia , Leptina/metabolismo , Leptina/farmacologia , Neurônios/efeitos dos fármacos , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Axônios/efeitos dos fármacos , Proteína Semelhante a ELAV 3/metabolismo , Receptor alfa de Estrogênio/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Integrases/genética , Integrases/metabolismo , Leptina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neuropeptídeo Y/genética , Neuropeptídeo Y/metabolismo , Receptores para Leptina/genética , Receptores para Leptina/metabolismo , Fator de Transcrição STAT3/metabolismoRESUMO
Following fasting, satiety is accompanied by neuronal activation in brain areas including the central amygdalar nucleus (CEA). Since CEA is known to inhibit food intake, we hypothesized that CEA contributes to the termination of meal during refeeding. To better understand the organization of this satiety-related circuit, the interconnections of the CEA with refeeding-activated neuronal groups were elucidated using retrograde (cholera toxin-ß subunit, CTB) and anterograde (phaseolus vulgaris leucoagglutinin, PHA-L) tracers in male rats. C-Fos-immunoreactivity was used as marker of neuronal activation. The refeeding-activated input of the CEA primarily originated from the paraventricular thalamic, parasubthalamic and parabrachial nuclei. Few CTB-c-Fos double-labeled neurons were detected in the prefrontal cortex, lateral hypothalamic area, nucleus of the solitary tract (NTS) and the bed nuclei of the stria terminalis (BNST). Only few refeeding-activated proopiomelanocortin-producing neurons of the arcuate nucleus projected to the CEA. Anterograde tract tracing revealed a high density of PHAL-labeled axons contacted with refeeding-activated neurons in the BNST, lateral hypothalamic area, parasubthalamic, paraventricular thalamic and parabrachial nuclei and NTS; a low density of labeled axons was found in the paraventricular hypothalamic nucleus. Chemogenetic activation of the medial CEA (CEAm) inhibited food intake during the first hour of refeeding, while activation of lateral CEA had no effect. These data demonstrate the existence of reciprocal connections between the CEA and distinct refeeding-activated hypothalamic, thalamic and brainstem nuclei, suggesting the importance of short feedback loops in the regulation of satiety and importance of the CEAm in the regulation of food intake during refeeding.
Assuntos
Mapeamento Encefálico , Núcleo Central da Amígdala/citologia , Núcleo Central da Amígdala/fisiologia , Vias Neurais/fisiologia , Neurônios/fisiologia , Resposta de Saciedade/fisiologia , Análise de Variância , Animais , Núcleo Arqueado do Hipotálamo/citologia , Núcleo Arqueado do Hipotálamo/fisiologia , Toxina da Cólera/metabolismo , Proteína Semelhante a ELAV 3/metabolismo , Ingestão de Alimentos/fisiologia , Jejum/fisiologia , Comportamento Alimentar/fisiologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Fito-Hemaglutininas/metabolismo , Pró-Opiomelanocortina/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Wistar , Transdução Genética , Proteína Vermelha FluorescenteRESUMO
Ran-binding protein family member, RanBP9 has been reported in various basic cellular mechanisms and neuropathological conditions including schizophrenia. Previous studies have reported that RanBP9 is highly expressed in the mammalian brain and retina; however, the role of RanBP9 in retinal development is largely unknown. Here, we present the novel and regulatory roles of RanBP9 in retinal development of a vertebrate animal model, zebrafish. Zebrafish embryos exhibited abundant expression of ranbp9 in developing brain tissues as well as in the developing retina. Yeast two-hybrid screening demonstrated the interaction of RanBP9 with Mind bomb, a component of Notch signaling involved in both neurogenesis and neural disease autism. The interaction is further substantiated by co-localization studies in cultured cells. Knockdown of ranbp9 resulted in retinal dysplasia with defective proliferation of retinal cells, downregulation of neuronal differentiation marker huC, elevation of neural proliferation marker her4, and alteration of cell cycle marker p57kip2. Expression of the Müller glial cell marker glutamine synthase was also affected in knockdown morphants. Our results suggest that Mind bomb-binding partner RanBP9 plays a role during retinal cell development of zebrafish embryogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Nucleares/metabolismo , Retina/embriologia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/metabolismo , Células COS , Proliferação de Células , Chlorocebus aethiops , Inibidor de Quinase Dependente de Ciclina p57/genética , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Proteínas do Citoesqueleto/genética , Regulação para Baixo , Proteína Semelhante a ELAV 3/genética , Proteína Semelhante a ELAV 3/metabolismo , Células Ependimogliais/fisiologia , Técnicas de Silenciamento de Genes , Neurogênese/fisiologia , Proteínas Nucleares/genética , Retina/citologia , Retina/metabolismo , Displasia Retiniana/genética , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases/genética , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genéticaRESUMO
The subthalamic nucleus (STN) is a main target structure of deep brain stimulation (DBS) in idiopathic Parkinson's disease. Nevertheless, there is an ongoing discussion regarding human STN volumes and neuron count, which could potentially have an impact on STN-DBS. Moreover, a suspected functional subdivision forms the basis of the tripartite hypothesis, which has not yet been morphologically substantiated. In this study, it was aimed to investigate the human STN by means of combined magnetic resonance imaging (MRI) and stereology. STN volumes were obtained from 14 individuals (ranging from 65 to 96 years, 25 hemispheres) in 3 T MRI and in luxol-stained histology slices. Neuron number and cell densities were investigated stereologically over the entire STN and in pre-defined subregions in anti-human neuronal protein HuC/D-stained slices. STN volumes measured with MRI were smaller than in stereology but appeared to be highly consistent, measuring on average 99 ± 6 mm3 (MRI) and 132 ± 20 mm3 (stereology). The neuron count was 431,088 ± 72,172. Both STN volumes and cell count decreased age-dependently. Neuron density was different for the dorsal, medial and ventral subregion with significantly higher values ventrally than dorsally. Small variations in STN volumes in both MRI and stereology contradict previous findings of large variations in STN size. Age-dependent decreases in STN volumes and neuron numbers might influence the efficacy of STN-DBS in a geriatric population. Though the study is limited in sample size, site-dependent differences for the STN subregions form a morphological basis for the tripartite theory. Hum Brain Mapp 38:909-922, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Envelhecimento , Imageamento por Ressonância Magnética , Técnicas Estereotáxicas , Núcleo Subtalâmico/citologia , Núcleo Subtalâmico/diagnóstico por imagem , Idoso , Idoso de 80 Anos ou mais , Contagem de Células , Proteína Semelhante a ELAV 3/metabolismo , Proteína Semelhante a ELAV 4/metabolismo , Feminino , Humanos , Imageamento Tridimensional , Masculino , Neurônios/metabolismoRESUMO
Glia play key roles in the regulation of neurotransmission in the nervous system. Fluoroacetate (FA) is a metabolic poison widely used to study glial functions by disrupting the tricarboxylic acid cycle enzyme aconitase. Despite the widespread use of FA, the effects of FA on essential glial functions such as calcium (Ca2+) signaling and hemichannel function remain unknown. Therefore, our goal was to assess specifically the impact of FA on essential glial cell functions that are involved with neurotransmission in the enteric nervous system. To this end, we generated a new optogenetic mouse model to study specifically the effects of FA on enteric glial Ca2+ signaling by crossing PC::G5-tdTomato mice with Sox10::creERT2 mice. FA did not change the peak glial Ca2+ response when averaged across all glia within a ganglion. However, FA decreased the percent of responding glia by 30% (P < 0.05) and increased the peak Ca2+ response of the glial cells that still exhibited a response by 26% (P < 0.01). Disruption of Ca2+ signaling with FA impaired the activity-dependent uptake of ethidium bromide through connexin-43 (Cx43) hemichannels (P < 0.05) but did not affect baseline Cx43-dependent dye uptake. FA did not cause overt glial or neurodegeneration, but glial cells significantly increased glial fibrillary acid protein by 56% (P < 0.05) following treatment with FA. Together, these data show that the acute impairment of glial metabolism with FA causes key changes in glial functions associated with their roles in neurotransmission and phenotypic changes indicative of reactive gliosis. NEW & NOTEWORTHY: Our study shows that the acute impairment of enteric glial metabolism with fluoroacetate (FA) alters specific glial functions that are associated with the modification of neurotransmission in the gut. These include subtle changes to glial agonist-evoked calcium signaling, the subsequent disruption of connexin-43 hemichannels, and changes in protein expression that are consistent with a transition to reactive glia. These changes in glial function offer a mechanistic explanation for the effects of FA on peripheral neuronal networks.