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1.
Mol Psychiatry ; 27(5): 2492-2501, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35296810

RESUMO

The global crisis of opioid overdose fatalities has led to an urgent search to discover the neurobiological mechanisms of opioid use disorder (OUD). A driving force for OUD is the dysphoric and emotionally painful state (hyperkatifeia) that is produced during acute and protracted opioid withdrawal. Here, we explored a mechanistic role for extrahypothalamic stress systems in driving opioid addiction. We found that glucocorticoid receptor (GR) antagonism with mifepristone reduced opioid addiction-like behaviors in rats and zebrafish of both sexes and decreased the firing of corticotropin-releasing factor neurons in the rat amygdala (i.e., a marker of brain stress system activation). In support of the hypothesized role of glucocorticoid transcriptional regulation of extrahypothalamic GRs in addiction-like behavior, an intra-amygdala infusion of an antisense oligonucleotide that blocked GR transcriptional activity reduced addiction-like behaviors. Finally, we identified transcriptional adaptations of GR signaling in the amygdala of humans with OUD. Thus, GRs, their coregulators, and downstream systems may represent viable therapeutic targets to treat the "stress side" of OUD.


Assuntos
Transtornos Relacionados ao Uso de Opioides , Síndrome de Abstinência a Substâncias , Corticosteroides , Animais , Hormônio Liberador da Corticotropina , Ratos , Peixe-Zebra
2.
J Nat Prod ; 84(4): 1232-1243, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33764053

RESUMO

Natural products such as conotoxins have tremendous potential as tools for biomedical research and for the treatment of different human diseases. Conotoxins are peptides present in the venoms of predatory cone snails that have a rich diversity of pharmacological functions. One of the major bottlenecks in natural products research is the rapid identification and evaluation of bioactive molecules. To overcome this limitation, we designed a set of light-induced behavioral assays in zebrafish larvae to screen for bioactive conotoxins. We used this screening approach to test several unique conotoxins derived from different cone snail clades and discovered that a conorfamide from Conus episcopatus, CNF-Ep1, had the most dramatic alterations in the locomotor behavior of zebrafish larvae. Interestingly, CNF-Ep1 is also bioactive in several mouse assay systems when tested in vitro and in vivo. Our novel screening platform can thus accelerate the identification of bioactive marine natural products, and the first compound discovered using this assay has intriguing properties that may uncover novel neuronal circuitry.


Assuntos
Larva/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Venenos de Moluscos/farmacologia , Neuropeptídeos/farmacologia , Peixe-Zebra , Animais , Caramujo Conus/química , Feminino , Masculino , Camundongos
3.
Mol Cell ; 50(2): 281-7, 2013 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-23541767

RESUMO

In metazoans, microRNAs play a critical role in the posttranscriptional regulation of genes required for cell proliferation and differentiation. MicroRNAs themselves are regulated by a multitude of mechanisms influencing their transcription and posttranscriptional maturation. However, there is only sparse knowledge on pathways regulating the mature, functional form of microRNA. Here, we uncover the implication of the decapping scavenger protein DCS-1 in the control of microRNA turnover. In Caenorhabditis elegans, mutations in dcs-1 increase the levels of functional microRNAs. We demonstrate that DCS-1 interacts with the exonuclease XRN-1 to promote microRNA degradation in an independent manner from its known decapping scavenger activity, establishing two molecular functions for DCS-1. Our findings thus indicate that DCS-1 is part of a degradation complex that performs microRNA turnover in animals.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimologia , MicroRNAs/metabolismo , N-Glicosil Hidrolases/metabolismo , RNA de Helmintos/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Exorribonucleases/metabolismo , Expressão Gênica , Mutação , N-Glicosil Hidrolases/genética , Pirofosfatases , Interferência de RNA , Estabilidade de RNA , Complexo de Inativação Induzido por RNA/metabolismo
4.
Nucleic Acids Res ; 45(3): 1488-1500, 2017 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-28180320

RESUMO

MiRNAs post-transcriptionally regulate gene expression by recruiting the miRNA-induced silencing complex (miRISC) to target mRNAs. However, the mechanisms by which miRISC components are maintained at appropriate levels for proper function are largely unknown. Here, we demonstrate that Caenorhabditis elegans TEG-1 regulates the stability of two miRISC effectors, VIG-1 and ALG-1, which in turn affects the abundance of miRNAs in various families. We demonstrate that TEG-1 physically interacts with VIG-1, and complexes with mature let-7 miRNA. Also, loss of teg-1 in vivo phenocopies heterochronic defects observed in let-7 mutants, suggesting the association of TEG-1 with miRISC is necessary for let-7 to function properly during development. Loss of TEG-1 function also affects the abundance and function of other microRNAs, suggesting that TEG-1's role is not specific to let-7. We further demonstrate that the human orthologs of TEG-1, VIG-1 and ALG-1 (CD2BP2, SERBP1/PAI-RBP1 and AGO2) are found in a complex in HeLa cells, and knockdown of CD2BP2 results in reduced miRNA levels; therefore, TEG-1's role in affecting miRNA levels and function is likely conserved. Together, these data demonstrate that TEG-1 CD2BP2 stabilizes miRISC and mature miRNAs, maintaining them at levels necessary to properly regulate target gene expression.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Modelos Biológicos , Mutação , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo
5.
PLoS Genet ; 9(11): e1003961, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24244204

RESUMO

Many core components of the microRNA pathway have been elucidated and knowledge of their mechanisms of action actively progresses. In contrast, factors with modulatory roles on the pathway are just starting to become known and understood. Using a genetic screen in Caenorhabditis elegans, we identify a component of the GARP (Golgi Associated Retrograde Protein) complex, vps-52, as a novel genetic interactor of the microRNA pathway. The loss of vps-52 in distinct sensitized genetic backgrounds induces the enhancement of defective microRNA-mediated gene silencing. It synergizes with the core microRNA components, alg-1 Argonaute and ain-1 (GW182), in enhancing seam cell defects and exacerbates the gene silencing defects of the let-7 family and lsy-6 microRNAs in the regulation of seam cell, vulva and ASEL neuron development. Underpinning the observed genetic interactions, we found that VPS-52 impinges on the abundance of the GW182 proteins as well as the levels of microRNAs including the let-7 family. Altogether, we demonstrate that GARP complex fulfills a positive modulatory role on microRNA function and postulate that acting through GARP, vps-52 participates in a membrane-related process of the microRNA pathway.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Proteínas de Transporte/genética , MicroRNAs/metabolismo , Proteínas de Transporte Vesicular/genética , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Transporte/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , MicroRNAs/genética , Interferência de RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
6.
J Clin Invest ; 131(10)2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-33848264

RESUMO

Opioid use disorder (OUD) has become a leading cause of death in the United States, yet current therapeutic strategies remain highly inadequate. To identify potential treatments for OUD, we screened a targeted selection of over 100 drugs using a recently developed opioid self-administration assay in zebrafish. This paradigm showed that finasteride, a steroidogenesis inhibitor approved for the treatment of benign prostatic hyperplasia and androgenetic alopecia, reduced self-administration of multiple opioids without affecting locomotion or feeding behavior. These findings were confirmed in rats; furthermore, finasteride reduced the physical signs associated with opioid withdrawal. In rat models of neuropathic pain, finasteride did not alter the antinociceptive effect of opioids and reduced withdrawal-induced hyperalgesia. Steroidomic analyses of the brains of fish treated with finasteride revealed a significant increase in dehydroepiandrosterone sulfate (DHEAS). Treatment with precursors of DHEAS reduced opioid self-administration in zebrafish in a fashion akin to the effects of finasteride. These results highlight the importance of steroidogenic pathways as a rich source of therapeutic targets for OUD and point to the potential of finasteride as a new treatment option for this disorder.


Assuntos
Inibidores de 5-alfa Redutase/farmacologia , Finasterida/farmacologia , Transtornos Relacionados ao Uso de Opioides/tratamento farmacológico , Animais , Modelos Animais de Doenças , Humanos , Masculino , Transtornos Relacionados ao Uso de Opioides/fisiopatologia , Ratos , Ratos Sprague-Dawley , Peixe-Zebra
7.
J Clin Invest ; 130(5): 2252-2269, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32202514

RESUMO

Prenatal alcohol exposure (PAE) affects at least 10% of newborns globally and leads to the development of fetal alcohol spectrum disorders (FASDs). Despite its high incidence, there is no consensus on the implications of PAE on metabolic disease risk in adults. Here, we describe a cohort of adults with FASDs that had an increased incidence of metabolic abnormalities, including type 2 diabetes, low HDL, high triglycerides, and female-specific overweight and obesity. Using a zebrafish model for PAE, we performed population studies to elucidate the metabolic disease seen in the clinical cohort. Embryonic alcohol exposure (EAE) in male zebrafish increased the propensity for diet-induced obesity and fasting hyperglycemia in adulthood. We identified several consequences of EAE that may contribute to these phenotypes, including a reduction in adult locomotor activity, alterations in visceral adipose tissue and hepatic development, and persistent diet-responsive transcriptional changes. Taken together, our findings define metabolic vulnerabilities due to EAE and provide evidence that behavioral changes and primary organ dysfunction contribute to resultant metabolic abnormalities.


Assuntos
Diabetes Mellitus Tipo 2 , Transtornos do Espectro Alcoólico Fetal , Obesidade , Efeitos Tardios da Exposição Pré-Natal , Adulto , Animais , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Feminino , Transtornos do Espectro Alcoólico Fetal/metabolismo , Transtornos do Espectro Alcoólico Fetal/patologia , Humanos , Recém-Nascido , Gordura Intra-Abdominal/metabolismo , Gordura Intra-Abdominal/patologia , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Transgênicos , Obesidade/etiologia , Obesidade/metabolismo , Obesidade/patologia , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Efeitos Tardios da Exposição Pré-Natal/patologia , Sistema de Registros , Peixe-Zebra
8.
Behav Brain Res ; 335: 158-166, 2017 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-28811180

RESUMO

The zebrafish (Danio rerio) has become an excellent tool to study mental health disorders, due to its physiological and genetic similarity to humans, ease of genetic manipulation, and feasibility of small molecule screening. Zebrafish have been shown to exhibit characteristics of addiction to drugs of abuse in non-contingent assays, including conditioned place preference, but contingent assays have been limited to a single assay for alcohol consumption. Using inexpensive electronic, mechanical, and optical components, we developed an automated opioid self-administration assay for zebrafish, enabling us to measure drug seeking and gain insight into the underlying biological pathways. Zebrafish trained in the assay for five days exhibited robust self-administration, which was dependent on the function of the µ-opioid receptor. In addition, a progressive ratio protocol was used to test conditioned animals for motivation. Furthermore, conditioned fish continued to seek the drug despite an adverse consequence and showed signs of stress and anxiety upon withdrawal of the drug. Finally, we validated our assay by confirming that self-administration in zebrafish is dependent on several of the same molecular pathways as in other animal models. Given the ease and throughput of this assay, it will enable identification of important biological pathways regulating drug seeking and could lead to the development of new therapeutic molecules to treat addiction.


Assuntos
Analgésicos Opioides/administração & dosagem , Comportamento de Procura de Droga , Peixe-Zebra , Animais , Comportamento Aditivo/etiologia , Comportamento Aditivo/psicologia , Comportamento Animal/efeitos dos fármacos , Condicionamento Clássico/efeitos dos fármacos , Modelos Animais de Doenças , Hidrocodona/administração & dosagem , Transtornos Relacionados ao Uso de Opioides/etiologia , Transtornos Relacionados ao Uso de Opioides/psicologia , Receptores Opioides mu/metabolismo , Autoadministração
9.
Sci Rep ; 5: 16688, 2015 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-26584588

RESUMO

The decapping scavenger enzyme DcpS is known for its role in hydrolyzing the cap structure following mRNA degradation. Recently, we discovered a new function in miRNA degradation activation for the ortholog of DcpS in C. elegans. Here we show that human DcpS conserves its role in miRNA turnover. In human cells, DcpS is a nucleocytoplasmic shuttling protein that activates miRNA degradation independently of its scavenger decapping activity in the cytoplasmic compartment. We also demonstrate that this new function for DcpS requires the contribution of the 5'-3' exonuclease Xrn2. Our findings support a conserved role of DcpS as a modulator of miRNA turnover in animals.


Assuntos
Núcleo Celular/metabolismo , Endorribonucleases/metabolismo , MicroRNAs/metabolismo , Capuzes de RNA/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Northern Blotting , Western Blotting , Endorribonucleases/genética , Imunofluorescência , Perfilação da Expressão Gênica/métodos , Células HEK293 , Humanos , MicroRNAs/genética , Dados de Sequência Molecular , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Capuzes de RNA/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos
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