Modulation of sleep behavior in zebrafish larvae by pharmacological targeting of the orexin receptor.

New pharmacological approaches that target orexin receptors (OXRs) are being developed to treat sleep disorders such as insomnia and narcolepsy, with fewer side effects than existing treatments. Orexins are neuropeptides that exert excitatory effects on postsynaptic neurons via the OXRs, and are important in regulating sleep/wake states. To date, there are three FDA-approved dual orexin receptor antagonists for the treatment of insomnia, and several small molecule oral OX2R (OXR type 2) agonists are in the pipeline for addressing the orexin deficiency in narcolepsy. To find new hypnotics and psychostimulants, rodents have been the model of choice, but they are costly and have substantially different sleep patterns to humans. As an alternative model, zebrafish larvae that like humans are diurnal and show peak daytime activity and rest at night offer several potential advantages including the ability for high throughput screening. To pharmacologically validate the use of a zebrafish model in the discovery of new compounds, we aimed in this study to evaluate the functionality of a set of known small molecule OX2R agonists and antagonists on human and zebrafish OXRs and to probe their effects on the behavior of zebrafish larvae. To this end, we developed an in vitro IP-One Homogeneous Time Resolved Fluorescence (HTRF) immunoassay, and in vivo locomotor assays that record the locomotor activity of zebrafish larvae under physiological light conditions as well as under dark-light triggers. We demonstrate that the functional IP-One test is a good predictor of biological activity in vivo. Moreover, the behavioral data show that a high-throughput assay that records the locomotor activity of zebrafish throughout the evening, night and morning is able to distinguish between OXR agonists and antagonists active on the zebrafish OXR. Conversely, a locomotor assay with alternating 30 min dark-light transitions throughout the day is not able to distinguish between the two sets of compounds, indicating the importance of circadian rhythm to their pharmacological activity. Overall, the results show that a functional IP-one test in combination with a behavioral assay using zebrafish is well-suited as a discovery platform to find novel compounds that target OXRs for the treatment of sleep disorders.

Hyperactivation of mTORC1 in a double hit mutant zebrafish model of tuberous sclerosis complex causes increased seizure susceptibility and neurodevelopmental abnormalities.

Tuberous sclerosis complex (TSC) is a multisystem genetic disorder caused by pathogenic variants in TSC1 and TSC2 genes. TSC patients present with seizures and brain abnormalities such as tubers and subependymal giant cells astrocytoma (SEGA). Despite common molecular and clinical features, the severity of the disease varies greatly, even intrafamilially. The second hit hypothesis suggests that an additional, inactivating mutation in the remaining functional allele causes a more severe phenotype and therefore explains the phenotypic variability. Recently, second hit mutations have been detected frequently in mTORopathies. To investigate the pathophysiological effects of second hit mutations, several mouse models have been developed. Here, we opted for a double mutant zebrafish model that carries a LOF mutation both in the tsc2 and the depdc5 gene. To the best of our knowledge, this is the first time a second-hit model has been studied in zebrafish. Significantly, the DEP domain-containing protein 5 (DEPDC5) gene has an important role in the regulation of mTORC1, and the combination of a germline TSC2 and somatic DEPDC5 mutation has been described in a TSC patient with intractable epilepsy. Our depdc5 -/- x tsc2 -/- double mutant zebrafish line displayed greatly increased levels of mammalian target of rapamycin (mTORC1) activity, augmented seizure susceptibility, and early lethality which could be rescued by rapamycin. Histological analysis of the brain revealed ventricular dilatation in the tsc2 and double homozygotes. RNA-sequencing showed a linear relation between the number of differentially expressed genes (DEGs) and the degree of mTORC1 hyperactivity. Enrichment analysis of their transcriptomes revealed that many genes associated with neurological developmental processes were downregulated and mitochondrial genes were upregulated. In particular, the transcriptome of human SEGA lesions overlapped strongly with the double homozygous zebrafish larvae. The data highlight the clinical relevance of the depdc5 -/- x tsc2 -/- double mutant zebrafish larvae that showed a more severe phenotype compared to the single mutants. Finally, analysis of gene-drug interactions identified interesting pharmacological targets for SEGA, underscoring the value of our small zebrafish vertebrate model for future drug discovery efforts.

Connectivity Mapping Using a Novel sv2a Loss-of-Function Zebrafish Epilepsy Model as a Powerful Strategy for Anti-epileptic Drug Discovery.

Synaptic vesicle glycoprotein 2A (SV2A) regulates action potential-dependent neurotransmitter release and is commonly known as the primary binding site of an approved anti-epileptic drug, levetiracetam. Although several rodent knockout models have demonstrated the importance of SV2A for functional neurotransmission, its precise physiological function and role in epilepsy pathophysiology remains to be elucidated. Here, we present a novel sv2a knockout model in zebrafish, a vertebrate with complementary advantages to rodents. We demonstrated that 6 days post fertilization homozygous sv2a-/- mutant zebrafish larvae, but not sv2a +/- and sv2a+/+ larvae, displayed locomotor hyperactivity and spontaneous epileptiform discharges, however, no major brain malformations could be observed. A partial rescue of this epileptiform brain activity could be observed after treatment with two commonly used anti-epileptic drugs, valproic acid and, surprisingly, levetiracetam. This observation indicated that additional targets, besides Sv2a, maybe are involved in the protective effects of levetiracetam against epileptic seizures. Furthermore, a transcriptome analysis provided insights into the neuropathological processes underlying the observed epileptic phenotype. While gene expression profiling revealed only one differentially expressed gene (DEG) between wildtype and sv2a +/- larvae, there were 4386 and 3535 DEGs between wildtype and sv2a-/- , and sv2a +/- and sv2a-/- larvae, respectively. Pathway and gene ontology (GO) enrichment analysis between wildtype and sv2a-/- larvae revealed several pathways and GO terms enriched amongst up- and down-regulated genes, including MAPK signaling, synaptic vesicle cycle, and extracellular matrix organization, all known to be involved in epileptogenesis and epilepsy. Importantly, we used the Connectivity map database to identify compounds with opposing gene signatures compared to the one observed in sv2a-/- larvae, to finally rescue the epileptic phenotype. Two out of three selected compounds rescued electrographic discharges in sv2a-/- larvae, while negative controls did not. Taken together, our results demonstrate that sv2a deficiency leads to increased seizure vulnerability and provide valuable insight into the functional importance of sv2a in the brain in general. Furthermore, we provided evidence that the concept of connectivity mapping represents an attractive and powerful approach in the discovery of novel compounds against epilepsy.

From the North Sea to Drug Repurposing, the Antiseizure Activity of Halimide and Plinabulin.

PharmaSea performed large-scale in vivo screening of marine natural product (MNP) extracts, using zebrafish embryos and larvae, to identify compounds with the potential to treat epilepsy. In this study, we report the discovery of two new antiseizure compounds, the 2,5-diketopiperazine halimide and its semi-synthetic analogue, plinabulin. Interestingly, these are both known microtubule destabilizing agents, and plinabulin could have the potential for drug repurposing, as it is already in clinical trials for the prevention of chemotherapy-induced neutropenia and treatment of non-small cell lung cancer. Both halimide and plinabulin were found to have antiseizure activity in the larval zebrafish pentylenetetrazole (PTZ) seizure model via automated locomotor analysis and non-invasive local field potential recordings. The efficacy of plinabulin was further characterized in animal models of drug-resistant seizures, i.e., the larval zebrafish ethyl ketopentenoate (EKP) seizure model and the mouse 6 Hz psychomotor seizure model. Plinabulin was observed to be highly effective against EKP-induced seizures, on the behavioral and electrophysiological level, and showed activity in the mouse model. These data suggest that plinabulin could be of interest for the treatment of drug-resistant seizures. Finally, the investigation of two functional analogues, colchicine and indibulin, which were observed to be inactive against EKP-induced seizures, suggests that microtubule depolymerization does not underpin plinabulin's antiseizure action.

Stress-induced inflammation evoked by immunogenic cell death is blunted by the IRE1α kinase inhibitor KIRA6 through HSP60 targeting.

Mounting evidence indicates that immunogenic therapies engaging the unfolded protein response (UPR) following endoplasmic reticulum (ER) stress favor proficient cancer cell-immune interactions, by stimulating the release of immunomodulatory/proinflammatory factors by stressed or dying cancer cells. UPR-driven transcription of proinflammatory cytokines/chemokines exert beneficial or detrimental effects on tumor growth and antitumor immunity, but the cell-autonomous machinery governing the cancer cell inflammatory output in response to immunogenic therapies remains poorly defined. Here, we profiled the transcriptome of cancer cells responding to immunogenic or weakly immunogenic treatments. Bioinformatics-driven pathway analysis indicated that immunogenic treatments instigated a NF-κB/AP-1-inflammatory stress response, which dissociated from both cell death and UPR. This stress-induced inflammation was specifically abolished by the IRE1α-kinase inhibitor KIRA6. Supernatants from immunogenic chemotherapy and KIRA6 co-treated cancer cells were deprived of proinflammatory/chemoattractant factors and failed to mobilize neutrophils and induce dendritic cell maturation. Furthermore, KIRA6 significantly reduced the in vivo vaccination potential of dying cancer cells responding to immunogenic chemotherapy. Mechanistically, we found that the anti-inflammatory effect of KIRA6 was still effective in IRE1α-deficient cells, indicating a hitherto unknown off-target effector of this IRE1α-kinase inhibitor. Generation of a KIRA6-clickable photoaffinity probe, mass spectrometry, and co-immunoprecipitation analysis identified cytosolic HSP60 as a KIRA6 off-target in the IKK-driven NF-κB pathway. In sum, our study unravels that HSP60 is a KIRA6-inhibitable upstream regulator of the NF-κB/AP-1-inflammatory stress responses evoked by immunogenic treatments. It also urges caution when interpreting the anti-inflammatory action of IRE1α chemical inhibitors.

Pericardial Injection of Kainic Acid Induces a Chronic Epileptic State in Larval Zebrafish.

Epilepsy is a common disorder of the brain characterized by spontaneous recurrent seizures, which develop gradually during a process called epileptogenesis. The mechanistic processes underlying the changes of brain tissue and networks toward increased seizure susceptibility are not fully understood. In rodents, injection of kainic acid (KA) ultimately leads to the development of spontaneous epileptic seizures, reflecting similar neuropathological characteristics as seen in patients with temporal lobe epilepsy (TLE). Although this model has significantly contributed to increased knowledge of epileptogenesis, it is technically demanding, costly to operate and hence not suitable for high-throughput screening of anti-epileptic drugs (AEDs). Zebrafish, a vertebrate with complementary advantages to rodents, is an established animal model for epilepsy research. Here, we generated a novel KA-induced epilepsy model in zebrafish larvae that we functionally and pharmacologically validated. KA was administered by pericardial injection at an early zebrafish larval stage. The epileptic phenotype induced was examined by quantification of seizure-like behavior using automated video recording, and of epileptiform brain activity measured via local field potential (LFP) recordings. We also assessed GFP-labeled GABAergic and RFP-labeled glutamatergic neurons in double transgenic KA-injected zebrafish larvae, and examined the GABA and glutamate levels in the larval heads by liquid chromatography with tandem mass spectrometry detection (LC-MS/MS). Finally, KA-injected larvae were exposed to five commonly used AEDs by immersion for pharmacological characterization of the model. Shortly after injection, KA induced a massive damage and inflammation in the zebrafish brain and seizure-like locomotor behavior. An abnormal reorganization of brain circuits was observed, a decrease in both GABAergic and glutamatergic neuronal population and their associated neurotransmitters. Importantly, these changes were accompanied by spontaneous and continuous epileptiform brain discharges starting after a short latency period, as seen in KA rodent models and reminiscent of human pathology. Three out of five AEDs tested rescued LFP abnormalities but did not affect the seizure-like behavior. Taken together, for the first time we describe a chemically-induced larval zebrafish epilepsy model offering unique insights into studying epileptogenic processes in vivo and suitable for high-throughput AED screening purposes and rapid genetic investigations.

Comparative Antiseizure Analysis of Diverse Natural Coumarin Derivatives in Zebrafish.

Coumarins are a well-known group of plant secondary metabolites with various pharmacological activities, including antiseizure activity. In the search for new antiseizure drugs (ASDs) to treat epilepsy, it is yet unclear which types of coumarins are particularly interesting as a systematic analysis has not been reported. The current study performed behavioral antiseizure activity screening of 18 different coumarin derivatives in the larval zebrafish pentylenetetrazole (PTZ) model using locomotor measurements. Activity was confirmed for seven compounds, which lowered seizure-like behavior as follows: oxypeucedanin 38%, oxypeucedanin hydrate 74%, notopterol 54%, nodakenetin 29%, hyuganin C 35%, daphnoretin 65%, and pimpinellin 60%. These coumarins, together with nodakenin, underwent further antiepileptiform analysis by local field potential recordings from the zebrafish opticum tectum (midbrain). All of them, except for nodakenetin, showed pronounced antiepileptiform activity, decreasing PTZ-induced elevation in power spectral density (PSD) by 83-89% for oxypeucedanin, oxypeucedanin hydrate, and notopterol, 77% for nodakenin, 26% for nodakenetin, 65% for hyuganin C, 88% for daphnoretin, and 81% for pimpinellin. These data demonstrate the potential of diverse coumarin scaffolds for ASD discovery. Finally, the structural differences between active and inactive coumarins were investigated in silico for oxypeucedanin hydrate and byacangelicin for their interaction with GABA-transaminase, a hypothetical target.

Modeling Neurodevelopmental Disorders and Epilepsy Caused by Loss of Function of kif2a in Zebrafish.

In recent years there has been extensive research on malformations of cortical development (MCDs) that result in clinical features like developmental delay, intellectual disability, and drug-resistant epilepsy (DRE). Various studies highlighted the contribution of microtubule-associated genes (including tubulin and kinesin encoding genes) in MCD development. It has been reported that de novo mutations in KIF2A, a member of the kinesin-13 family, are linked to brain malformations and DRE. Although it is known that KIF2A functions by regulating microtubule depolymerization via an ATP-driven process, in vivo implications of KIF2A loss of function remain partly unclear. Here, we present a novel kif2a knock-out zebrafish model, showing hypoactivity, habituation deficits, pentylenetetrazole-induced seizure susceptibility and microcephaly, as well as neuronal cell proliferation defects and increased apoptosis. Interestingly, kif2a-/- larvae survived until adulthood and were fertile. Notably, our kif2a zebrafish knock-out model demonstrated many phenotypic similarities to KIF2A mouse models. This study provides valuable insights into the functional importance of kif2a in zebrafish and phenotypical hallmarks related to KIF2A mutations. Ultimately, this model could be used in a future search for more effective therapies that alleviate the clinical symptoms typically associated with MCDs.

An Emerging Role for Sigma-1 Receptors in the Treatment of Developmental and Epileptic Encephalopathies.

Developmental and epileptic encephalopathies (DEEs) are complex conditions characterized primarily by seizures associated with neurodevelopmental and motor deficits. Recent evidence supports sigma-1 receptor modulation in both neuroprotection and antiseizure activity, suggesting that sigma-1 receptors may play a role in the pathogenesis of DEEs, and that targeting this receptor has the potential to positively impact both seizures and non-seizure outcomes in these disorders. Recent studies have demonstrated that the antiseizure medication fenfluramine, a serotonin-releasing drug that also acts as a positive modulator of sigma-1 receptors, reduces seizures and improves everyday executive functions (behavior, emotions, cognition) in patients with Dravet syndrome and Lennox-Gastaut syndrome. Here, we review the evidence for sigma-1 activity in reducing seizure frequency and promoting neuroprotection in the context of DEE pathophysiology and clinical presentation, using fenfluramine as a case example. Challenges and opportunities for future research include developing appropriate models for evaluating sigma-1 receptors in these syndromic epileptic conditions with multisystem involvement and complex clinical presentation.

Pharmacokinetics in Zebrafish Embryos (ZFE) Following Immersion and Intrayolk Administration: A Fluorescence-Based Analysis.

Zebrafish embryos (ZFE) have increasingly gained in popularity as a model to perform safety screenings of compounds. Although immersion of ZFE is the main route of exposure used, evidence shows that not all small molecules are equally absorbed, possibly resulting in false-negative readouts and incorrect conclusions. In this study, we compared the pharmacokinetics of seven fluorescent compounds with known physicochemical properties that were administered to two-cell stage embryos by immersion or by IY microinjection. Absorption and distribution of the dyes were followed at various timepoints up to 120 hpf by spatiotemporal fluorescence imaging. The concentration (10 µM) and dose (2 mg/kg) used were selected as quantities typically applied in preclinical experiments and zebrafish studies. The data show that in the case of a lipophilic compound (log D: 1.73) the immersion procedure resulted in an intrabody exposure which is similar or higher than that seen after the IY microinjection. In contrast, zero to low intrabody exposure was reached after immersion of the embryos with less lipophilic compounds. In the latter case IY microinjection, a technical procedure that can be easily automated, is highly recommended.

Pre-validation of choriogenin H transgenic medaka eleutheroembryos as a quantitative estrogenic activity test method.

The choriogenin H - EGFP transgenic medaka (Oryzias melastigma) has been used to test estrogenic substances and quantify estrogenic activity into 17ß-estradiol (E2) equivalency (EEQ). The method uses 8 eleutheroembryos in 2 ml solution per well and 3 wells per treatment in 24-well plates at 26 ± 1 °C for 24 ± 2 h, with subsequent measurements of induced GFP signal intensity. EEQ measurements are calculated using a E2 probit regression model with a coefficient of determination (R2) > 0.90. The selectivity was confirmed evaluating 27 known estrogenic and 5 known non-estrogenic compounds. Limit of quantitation (LOQ), recovery rate and bias were calculated to be 1 ng/ml EEQ, 104% and 4% respectively. Robustness analysis revealed exposure temperature is a sensitive parameter that should be kept at 26 ± 1 °C. The repeatability of intra- and inter-laboratories achieved CV < 30% for most tested food and cosmetics samples. The lot-lot stability was confirmed by the stable EEQ qualitative control (QC, 1 ng/mL E2) and calibration curve results. The stability of standard reagents, samples and sample extracts was also investigated. These data demonstrated this method to be an accurate indicator of estrogenic activity for both chemicals and extracts.

Using Zebrafish as a Disease Model to Study Fibrotic Disease.

In drug discovery, often animal models are used that mimic human diseases as closely as possible. These animal models can be used to address various scientific questions, such as testing and evaluation of new drugs, as well as understanding the pathogenesis of diseases. Currently, the most commonly used animal models in the field of fibrosis are rodents. Unfortunately, rodent models of fibrotic disease are costly and time-consuming to generate. In addition, present models are not very suitable for screening large compounds libraries. To overcome these limitations, there is a need for new in vivo models. Zebrafish has become an attractive animal model for preclinical studies. An expanding number of zebrafish models of human disease have been documented, for both acute and chronic diseases. A deeper understanding of the occurrence of fibrosis in zebrafish will contribute to the development of new and potentially improved animal models for drug discovery. These zebrafish models of fibrotic disease include, among others, cardiovascular disease models, liver disease models (categorized into Alcoholic Liver Diseases (ALD) and Non-Alcoholic Liver Disease (NALD)), and chronic pancreatitis models. In this review, we give a comprehensive overview of the usage of zebrafish models in fibrotic disease studies, highlighting their potential for high-throughput drug discovery and current technical challenges.

Efficacy of Fenfluramine and Norfenfluramine Enantiomers and Various Antiepileptic Drugs in a Zebrafish Model of Dravet Syndrome.

Dravet syndrome (DS) is a rare genetic encephalopathy that is characterized by severe seizures and highly resistant to commonly used antiepileptic drugs (AEDs). In 2020, FDA has approved fenfluramine (FFA) for treatment of seizures associated with DS. However, the clinically used FFA is a racemic mixture (i.e. (±)-FFA), that is substantially metabolized to norfenfluramine (norFFA), and it is presently not known whether the efficacy of FFA is due to a single enantiomer of FFA, or to both, and whether the norFFA enantiomers also contribute significantly. In this study, the antiepileptic activity of enantiomers of FFA (i.e. (+)-FFA and (-)-FFA) and norFFA (i.e. (+)-norFFA and (-)-norFFA) was explored using the zebrafish scn1Lab-/- mutant model of DS. To validate the experimental conditions used, we assessed the activity of various AEDs typically used in the fight against DS, including combination therapy. Overall, our results are highly consistent with the treatment algorithm proposed by the updated current practice in the clinical management of DS. Our results show that (+)-FFA, (-)-FFA and (+)-norFFA displayed significant antiepileptic effects in the preclinical model, and thus can be considered as compounds actively contributing to the clinical efficacy of FFA. In case of (-)-norFFA, the results were less conclusive. We also investigated the uptake kinetics of the enantiomers of FFA and norFFA in larval zebrafish heads. The data show that the total uptake of each compound increased in a time-dependent fashion. A somewhat similar uptake was observed for the (+)-norFFA and (-)-norFFA, implying that the levo/dextrotation of the structure did not dramatically affect the uptake. Significantly, when comparing (+)-FFA with the less lipophilic (+)-norFFA, the data clearly show that the nor-metabolite of FFA is taken up less than the parent compound.

Nephrotoxic Effects in Zebrafish after Prolonged Exposure to Aristolochic Acid.

With the aim to explore the possibility to generate a zebrafish model of renal fibrosis, in this study the fibrogenic renal effect of aristolochic acid I (AAI) after immersion was assessed. This compound is highly nephrotoxic able to elicit renal fibrosis after exposure of rats and humans. Our results reveal that larval zebrafish at 15 days dpf (days post-fertilization) exposed for 8 days to 0.5 µM AAI showed clear signs of AKI (acute kidney injury). The damage resulted in the relative loss of the functional glomerular filtration barrier. Conversely, we did not observe any deposition of collagen, nor could we immunodetect α-SMA, a hallmark of myofibroblasts, in the tubules. In addition, no increase in gene expression of fibrogenesis biomarkers after whole animal RNA extraction was found. As zebrafish have a high capability for tissue regeneration possibly impeding fibrogenic processes, we also used a tert-/- zebrafish line exhibiting telomerase deficiency and impaired tissue homeostasis. AAI-treated tert-/- larvae displayed an increased sensitivity towards 0.5 µM AAI. Importantly, after AAI treatment a mild collagen deposition could be found in the tubules. The outcome implies that sustained AKI induced by nephrotoxic compounds combined with defective tert-/- stem cells can produce a fibrotic response.

Fenfluramine acts as a positive modulator of sigma-1 receptors.

OBJECTIVE: Adjunctive fenfluramine hydrochloride, classically described as acting pharmacologically through a serotonergic mechanism, has demonstrated a unique and robust clinical response profile with regard to its magnitude, consistency, and durability of effect on seizure activity in patients with pharmacoresistant Dravet syndrome. Recent findings also support long-term improvements in executive functions (behavior, emotion, cognition) in these patients. The observed clinical profile is inconsistent with serotonergic activity alone, as other serotonergic medications have not been demonstrated to have these clinical effects. This study investigated a potential role for σ1 receptor activity in complementing fenfluramine's serotonergic pharmacology. METHODS: Radioligand binding assays tested the affinity of fenfluramine for 47 receptors associated with seizures in the literature, including σ receptors. Cellular function assays tested fenfluramine and norfenfluramine (its major metabolite) activity at various receptors, including adrenergic, muscarinic, and serotonergic receptors. The σ1 receptor activity was assessed by the mouse vas deferens isometric twitch and by an assay of dissociation of the σ1 receptor from the endoplasmic reticulum stress protein binding immunoglobulin protein (BiP). In vivo mouse models assessed fenfluramine activity at σ1 receptors in ameliorating dizocilpine-induced learning deficits in spatial and nonspatial memory tasks, alone or in combination with the reference σ1 receptor agonist PRE-084. RESULTS: Fenfluramine and norfenfluramine bound ≥30% to ß2-adrenergic, muscarinic M1, serotonergic 5-HT1A, and σ receptors, as well as sodium channels, with a Ki between 266 nM (σ receptors) and 17.5 µM (ß-adrenergic receptors). However, only σ1 receptor isometric twitch assays showed a positive functional response, with weak stimulation by fenfluramine and inhibition by norfenfluramine. Fenfluramine, but not the 5-HT2C agonist lorcaserin, showed a positive modulation of the PRE-084-induced dissociation of σ1 protein from BiP. Fenfluramine also showed dose-dependent antiamnesic effects against dizocilpine-induced learning deficits in spontaneous alternation and passive avoidance responses, which are models of σ1 activation. Moreover, low doses of fenfluramine synergistically potentiated the low-dose effect of PRE-084, confirming a positive modulatory effect at the σ1 receptor. Finally, all in vivo effects were blocked by the σ1 receptor antagonist NE-100. SIGNIFICANCE: Fenfluramine demonstrated modulatory activity at σ1 receptors in vitro and in vivo in addition to its known serotonergic activity. These studies identify a possible new σ1 receptor mechanism underpinning fenfluramine's central nervous system effects, which may contribute to its antiseizure activity in Dravet syndrome and positive effects observed on executive functions in clinical studies.

Zebrafish-Based Discovery of Antiseizure Compounds from the North Sea: Isoquinoline Alkaloids TMC-120A and TMC-120B.

There is a high need for the development of new and improved antiseizure drugs (ASDs) to treat epilepsy. Despite the potential of marine natural products (MNPs), the EU marine biodiscovery consortium PharmaSea has made the only effort to date to perform ASD discovery based on large-scale screening of MNPs. To this end, the embryonic zebrafish photomotor response assay and the larval zebrafish pentylenetetrazole (PTZ) model were used to screen MNP extracts for neuroactivity and antiseizure activity, respectively. Here we report the identification of the two known isoquinoline alkaloids TMC-120A and TMC-120B as novel antiseizure compounds, which were isolated by bioactivity-guided purification from the marine-derived fungus Aspergillus insuetus. TMC-120A and TMC-120B were observed to significantly lower PTZ-induced seizures and epileptiform brain activity in the larval zebrafish PTZ seizure model. In addition, their structural analogues TMC-120C, penicisochroman G, and ustusorane B were isolated and also significantly lowered PTZ-induced seizures. Finally, TMC-120A and TMC-120B were investigated in a mouse model of drug-resistant focal seizures. Compound treatment significantly shortened the seizure duration, thereby confirming their antiseizure activity. These data underscore the possibility to translate findings in zebrafish to mice in the field of epilepsy and the potential of the marine environment for ASD discovery.

Cannabidiol modulates phosphorylated rpS6 signalling in a zebrafish model of Tuberous Sclerosis Complex.

Tuberous sclerosis complex (TSC) is a rare disease caused by mutations in the TSC1 or TSC2 genes and is characterized by widespread tumour growth, intractable epilepsy, cognitive deficits and autistic behaviour. CBD has been reported to decrease seizures and inhibit tumour cell progression, therefore we sought to determine the influence of CBD on TSC pathology in zebrafish carrying a nonsense mutation in the tsc2 gene. CBD treatment from 6 to 7 days post-fertilization (dpf) induced significant anxiolytic actions without causing sedation. Furthermore, CBD treatment from 3 dpf had no impact on tsc2-/- larvae motility nor their survival. CBD treatment did, however, reduce the number of phosphorylated rpS6 positive cells, and their cross-sectional cell size. This suggests a CBD mediated suppression of mechanistic target of rapamycin (mTOR) activity in the tsc2-/- larval brain. Taken together, these data suggest that CBD selectively modulates levels of phosphorylated rpS6 in the brain and additionally provides an anxiolytic effect. This is pertinent given the alterations in mTOR signalling in experimental models of TSC. Additional work is necessary to identify upstream signal modulation and to further justify the use of CBD as a possible therapeutic strategy to manage TSC.

Identification of GSK-3 as a Potential Therapeutic Entry Point for Epilepsy.

In view of the clinical need for new antiseizure drugs (ASDs) with novel modes of action, we used a zebrafish seizure model to screen the anticonvulsant activity of medicinal plants used by traditional healers in the Congo for the treatment of epilepsy, and identified a crude plant extract that inhibited pentylenetetrazol (PTZ)-induced seizures in zebrafish larvae. Zebrafish bioassay-guided fractionation of this anticonvulsant Fabaceae species, Indigofera arrecta, identified indirubin, a compound with known inhibitory activity of glycogen synthase kinase (GSK)-3, as the bioactive component. Indirubin, as well as the more potent and selective GSK-3 inhibitor 6-bromoindirubin-3'-oxime (BIO-acetoxime) were tested in zebrafish and rodent seizure assays. Both compounds revealed anticonvulsant activity in PTZ-treated zebrafish larvae, with electroencephalographic recordings revealing reduction of epileptiform discharges. Both indirubin and BIO-acetoxime also showed anticonvulsant activity in the pilocarpine rat model for limbic seizures and in the 6-Hz refractory seizure mouse model. Most interestingly, BIO-acetoxime also exhibited anticonvulsant actions in 6-Hz fully kindled mice. Our findings thus provide the first evidence for anticonvulsant activity of GSK-3 inhibition, thereby implicating GSK-3 as a potential therapeutic entry point for epilepsy. Our results also support the use of zebrafish bioassay-guided fractionation of antiepileptic medicinal plant extracts as an effective strategy for the discovery of new ASDs with novel mechanisms of action.

Bioassay-guided isolation of anti-seizure principles from Semen Pharbitidis using a zebrafish pentylenetetrazol seizure model.

ETHNOPHARMACOLOGICAL RELEVANCE: Semen Pharbitidis, the seeds of Pharbitis nil (Linn.) Choisy (Convolvulaceae) is a well-known traditional Chinese medicinal plant used for treating helminthiasis and epilepsy in China. AIM OF THE STUDY: This study aims to identify the anti-seizure components from Semen Pharbitidis. METHODS: A bioassay-guided isolation of anti-seizure compounds from Semen Pharbitidis was performed using a zebrafish pentylenetetrazol seizure model. The structures of active compounds were elucidated by high resolution mass spectrometry. The fragments of active compounds were tested for anti-seizure activity as well. RESULTS: The bioassay-guided isolation of ethanol extract of Semen Pharbitidis led to a group of resin glucosides, namely pharbitin. One of the fragments of pharbitin, 2-methylbutyric acid, also showed anti-seizure activity. CONCLUSIONS: We provided further experimental scientific evidence to support the traditional use of Semen Pharbitidis for the treatment of epilepsy. Pharbitin was identified to be the main anti-seizure component in Semen Pharbitidis.

Genetic Renal Diseases: The Emerging Role of Zebrafish Models.

The structural and functional similarity of the larval zebrafish pronephros to the human nephron, together with the recent development of easier and more precise techniques to manipulate the zebrafish genome have motivated many researchers to model human renal diseases in the zebrafish. Over the last few years, great advances have been made, not only in the modeling techniques of genetic diseases in the zebrafish, but also in how to validate and exploit these models, crossing the bridge towards more informative explanations of disease pathophysiology and better designed therapeutic interventions in a cost-effective in vivo system. Here, we review the significant progress in these areas giving special attention to the renal phenotype evaluation techniques. We further discuss the future applications of such models, particularly their role in revealing new genetic diseases of the kidney and their potential use in personalized medicine.