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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.

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.

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.

Opioid use disorder (OUD) has become a leading cause of death in the US, yet current therapeutic strategies remain highly inadequate. To identify novel 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 did not interfere with the antinociceptive effect of opioids in rat models of neuropathic pain. 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. Our 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 option for this disorder.

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.

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.

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.

A new twist in the microRNA pathway: Not Dicer but Argonaute is required for a microRNA production

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., Author Summary The microRNA pathway is a post-transcriptional gene regulatory system that uses small non-coding RNAs called microRNAs to control multiple developmental and physiological processes. With the goal of unveiling factors modulating this regulatory pathway, we have undertaken the exploration of genetic interactors in the roundworm Caenorhabditis elegans. We identify vps-52, a component of the Golgi Associated Retrograde Protein or GARP complex, and establish that this complex executes a positive modulatory role on microRNA activity. The absence of vps-52 function exacerbates diverse microRNA-related defects. Molecularly, this effect relates to decreased abundance of microRNAs and the GW182 proteins. Considering that GARP is involved in the traffic of vesicles from endosomes back to the Golgi apparatus, we propose that GARP facilitates a membrane-related process of the microRNA pathway.

In metazoans, microRNAs play a critical role in the post-transcriptional regulation of genes required for cell proliferation and differentiation. microRNAs themselves are regulated by a multitude of mechanisms influencing their transcription and post-transcriptional maturation. However, there is only sparse knowledge on pathways regulating the mature, functional form of a microRNA. Here, we identify a new player in the control of microRNA turnover, the decapping scavenger protein DCS-1. In Caenorhabditis elegans, mutations in dcs-1 increase the levels of functional microRNAs. We demonstrate that DCS-1 interacts with the exonuclease XRN-1 in vivo and in vitro, that this interaction promotes microRNA degradation and that this new function does not require its decapping scavenger activity, establishing independence of these two processes. Our findings thus indicate that DCS-1 is part of a degradation complex that performs microRNA turnover in animals.