Cyberlindnera jadinii yeast as a functional protein source: Modulation of immunoregulatory pathways in the intestinal proteome of zebrafish (Danio rerio).

Yeasts contain bioactive components that can enhance fish immune robustness and disease resistance. Our study focused on analyzing intestinal immunoregulatory pathways in zebrafish (Danio rerio) using iTRAQ and 2D LC-MS/MS to quantify intestinal proteins. Zebrafish were fed either control diet (C) or diet supplemented with autolyzed Cyberlindnera jadinii (ACJ). KEGG analysis revealed that ACJ yeast diet induced increased abundance of proteins related to arginine and proline metabolism, phagosome, C-lectin receptor signaling, ribosome and PPAR signaling pathways, which can modulate and enhance innate immune responses. ACJ yeast diet also showed decreased abundance of proteins associated with inflammatory pathways, including apoptosis, necroptosis and ferroptosis. These findings indicate boosted innate immune response and control of inflammation-related pathways in zebrafish intestine. Our findings in the well annotated proteome of zebrafish enabled a detailed investigation of intestinal responses and provide insight into health-beneficial effects of yeast species C. jadinii, which is relevant for aquaculture species.

CLN3 deficiency leads to neurological and metabolic perturbations during early development.

Juvenile neuronal ceroid lipofuscinosis (or Batten disease) is an autosomal recessive, rare neurodegenerative disorder that affects mainly children above the age of 5 yr and is most commonly caused by mutations in the highly conserved CLN3 gene. Here, we generated cln3 morphants and stable mutant lines in zebrafish. Although neither morphant nor mutant cln3 larvae showed any obvious developmental or morphological defects, behavioral phenotyping of the mutant larvae revealed hyposensitivity to abrupt light changes and hypersensitivity to pro-convulsive drugs. Importantly, in-depth metabolomics and lipidomics analyses revealed significant accumulation of several glycerophosphodiesters (GPDs) and cholesteryl esters, and a global decrease in bis(monoacylglycero)phosphate species, two of which (GPDs and bis(monoacylglycero)phosphates) were previously proposed as potential biomarkers for CLN3 disease based on independent studies in other organisms. We could also demonstrate GPD accumulation in human-induced pluripotent stem cell-derived cerebral organoids carrying a pathogenic variant for CLN3 Our models revealed that GPDs accumulate at very early stages of life in the absence of functional CLN3 and highlight glycerophosphoinositol and BMP as promising biomarker candidates for pre-symptomatic CLN3 disease.

Seizure-induced increase in microglial cell population in the developing zebrafish brain.

Epilepsy is a chronic brain disorder characterized by unprovoked and recurrent seizures, of which 60% are of unknown etiology. Recent studies implicate microglia in the pathophysiology of epilepsy. However, their role in this process, in particular following early-life seizures, remains poorly understood due in part to the lack of suitable experimental models allowing the in vivo imaging of microglial activity. Given the advantage of zebrafish larvae for minimally-invasive imaging approaches, we sought for the first time to describe the microglial responses after acute seizures in two different zebrafish larval models: a chemically-induced epileptic model by the systemic injection of kainate at 3 days post-fertilization, and the didys552 genetic epilepsy model, which carries a mutation in scn1lab that leads to spontaneous epileptiform discharges. Kainate-treated larvae exhibited transient brain damage as shown by increased numbers of apoptotic nuclei as early as one day post-injection, which was followed by an increase in the number of microglia in the brain. A similar microglial phenotype was also observed in didys552-/- mutants, suggesting that microglia numbers change in response to seizure-like activity in the brain. Interestingly, kainate-treated larvae also displayed a decreased seizure threshold towards subsequent pentylenetetrazole-induced seizures, as shown by higher locomotor and encephalographic activity in comparison with vehicle-injected larvae. These results are comparable to kainate-induced rodent seizure models and suggest the suitability of these zebrafish seizure models for future studies, in particular to elucidate the links between epileptogenesis and microglial dynamic changes after seizure induction in the developing brain, and to understand how these modulate seizure susceptibility.

Natural product-derived therapies for treating drug-resistant epilepsies: From ethnopharmacology to evidence-based medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: Epilepsy is one of the most prevalent neurological human diseases, affecting 1% of the population in all age groups. Despite the availability of over 25 anti-seizure medications (ASMs), which are approved in most industrialized countries, approximately 30% of epilepsy patients still experience seizures that are resistant to these drugs. Since ASMs target only limited number of neurochemical mechanisms, drug-resistant epilepsy (DRE) is not only an unmet medical need, but also a formidable challenge in drug discovery. AIM: In this review, we examine recently approved epilepsy drugs based on natural product (NP) such as cannabidiol (CBD) and rapamycin, as well as NP-based epilepsy drug candidates still in clinical development, such as huperzine A. We also critically evaluate the therapeutic potential of botanical drugs as polytherapy or adjunct therapy specifically for DRE. METHODS: Articles related to ethnopharmacological anti-epileptic medicines and NPs in treating all forms of epilepsy were collected from PubMed and Scopus using keywords related to epilepsy, DRE, herbal medicines, and NPs. The database clinicaltrials.gov was used to find ongoing, terminated and planned clinical trials using herbal medicines or NPs in epilepsy treatment. RESULTS: A comprehensive review on anti-epileptic herbal drugs and natural products from the ethnomedical literature is provided. We discuss the ethnomedical context of recently approved drugs and drug candidates derived from NPs, including CBD, rapamycin, and huperzine A. Recently published studies on natural products with preclinical efficacy in animal models of DRE are summarized. Moreover, we highlight that natural products capable of pharmacologically activating the vagus nerve (VN), such as CBD, may be therapeutically useful to treat DRE. CONCLUSIONS: The review highlights that herbal drugs utilized in traditional medicine offer a valuable source of potential anti-epileptic drug candidates with novel mechanisms of action, and with clinical promise for the treatment of drug-resistant epilepsy (DRE). Moreover, recently developed NP-based anti-seizure medications (ASMs) indicate the translational potential of metabolites of plant, microbial, fungal and animal origin.

Australindolones, New Aminopyrimidine Substituted Indolone Alkaloids from an Antarctic Tunicate Synoicum sp.

Five new alkaloids have been isolated from the lipophilic extract of the Antarctic tunicate Synoicum sp. Deep-sea specimens of Synoicum sp. were collected during a 2011 cruise of the R/V Nathanial B. Palmer to the southern Scotia Arc, Antarctica. Crude extracts from the invertebrates obtained during the cruise were screened in a zebrafish-based phenotypic assay. The Synoicum sp. extract induced embryonic dysmorphology characterized by axis truncation, leading to the isolation of aminopyrimidine substituted indolone (1-4) and indole (5-12) alkaloids. While the primary bioactivity tracked with previously reported meridianins A-G (5-11), further investigation resulted in the isolation and characterization of australindolones A-D (1-4) and the previously unreported meridianin H (12).

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.

Evaluation of the wound healing properties of South African medicinal plants using zebrafish and in vitro bioassays.

ETHNOPHARMACOLOGICAL RELEVANCE: In South Africa, medicinal plants have a history of traditional use, with many species used for treating wounds. The scientific basis of such uses remains largely unexplored. AIM OF THE STUDY: To screen South African plants used ethnomedicinally for wound healing based on their pro-angiogenic and wound healing activity, using transgenic zebrafish larvae and cell culture assays. MATERIALS AND METHODS: South African medicinal plants used for wound healing were chosen according to literature. Dried plant material was extracted using six solvents of varying polarities. Pro-angiogenesis was assessed in vivo by observing morphological changes in sub-intestinal vessels after crude extract treatment of transgenic zebrafish larvae with vasculature-specific expression of a green fluorescent protein. Subsequently, the in vitro anti-inflammatory, fibroblast proliferation and collagen production effects of the plant extracts that were active in the zebrafish angiogenesis assay were investigated using murine macrophage (RAW 264.7) and human fibroblast (MRHF) cell lines. RESULTS: Fourteen plants were extracted using six different solvents to yield 84 extracts and the non-toxic (n=72) were initially screened for pro-angiogenic activity in the zebrafish assay. Of these plant species, extracts of Lobostemon fruticosus, Scabiosa columbaria and Cotyledon orbiculata exhibited good activity in a concentration-dependent manner. All active extracts showed negligible in vitro toxicity using the MTT assay. Lobostemon fruticosus and Scabiosa columbaria extracts showed noteworthy anti-inflammatory activity in RAW 264.7 macrophages. The acetone extract of Lobostemon fruticosus stimulated the most collagen production at 122% above control values using the MRHF cell line, while all four of the selected extracts significantly stimulated cellular proliferation in vitro in the MRHF cell line. CONCLUSIONS: The screening of the selected plant species provided valuable preliminary information validating the use of some of the plants in traditional medicine used for wound healing in South Africa. This study is the first to discover through an evidence-based pharmacology approach the wound healing properties of such plant species using the zebrafish as an in vivo model.

Anti-seizure activity of African medicinal plants: The identification of bioactive alkaloids from the stem bark of Rauvolfia caffra using an in vivo zebrafish model.

ETHNOPHARMACOLOGICAL RELEVANCE: Epilepsy is one of the major chronic diseases that does not have a cure to date. Adverse drug reactions have been reported from the use of available anti-epileptic drugs (AEDs) which are also effective in only two-thirds of the patients. Accordingly, the identification of scaffolds with promising anti-seizure activity remains an important first step towards the development of new anti-epileptic therapies, with improved efficacy and reduced adverse effects. Herbal medicines are widely used in developing countries, including in the treatment of epilepsy but with little scientific evidence to validate this use. In the search for new epilepsy treatment options, the zebrafish has emerged as a chemoconvulsant-based model for epilepsy, mainly because of the many advantages that zebrafish larvae offer making them highly suitable for high-throughput drug screening. AIM OF THE STUDY: In this study, 20 medicinal plants traditionally used in South Africa to treat epilepsy were screened for anti-epileptic activity using a zebrafish larvae model. MATERIALS AND METHODS: Toxicity triaging was conducted on 120 crude extracts, 44 fractions and three isolated compounds to determine the maximum tolerated concentration (MTC) of each extract, fraction or compound. MTC values were used to guide the concentration range selection in bioactivity studies. The effectiveness of crude extracts, fractions and isolated compounds from Rauvolfia caffra Sond. in suppression of pentylenetetrazole (PTZ) induced seizure-like behaviour in a 6-dpf zebrafish larvae model was measured using the PTZ assay. RESULTS: Following a preliminary toxicity triage and bioactivity screen of crude extracts from 20 African plants used traditionally for the treatment and management of epilepsy, the methanolic extract of Rauvolfia caffra Sond. was identified as the most promising at suppressing PTZ induced seizure-like behaviour in a zebrafish larvae model. Subsequent bioactivity-guided fractionation and spectroscopic structural elucidation resulted in the isolation and identification of two tryptoline derivatives; a previously unreported alkaloid to which we assigned the trivial name rauverine H (1) and the known alkaloid pleiocarpamine (2). Pleiocarpamine was found to reduce PTZ-induced seizures in a dose-dependent manner. CONCLUSIONS: Accordingly, pleiocarpamine represents a promising scaffold for the development of new anti-seizure therapeutic compounds. Furthermore, the results of this study provide preliminary evidence to support the traditional use of Rauvolfia caffra Sond. in the treatment and management of epilepsy. These findings warrant further studies on the anti-epileptic potential of Rauvolfia caffra Sond. using other models.

Identification of Potential Antiseizure Agents in Boswellia sacra using In Vivo Zebrafish and Mouse Epilepsy Models.

The resin of the tree Boswellia sacra Flueck. (synonym: B. carterii; Burseraceae), also known as "frankincense", is a traditional remedy used for central nervous system disorders in East Africa. Here we report the evaluation of its antiseizure activity in zebrafish and mouse epilepsy models to identify novel antiseizure compounds. The resin was extracted by solvents of increasing polarity. The hexane extract demonstrated the strongest antiseizure activity and was therefore subjected to bioactivity-guided isolation, which leaded to the isolation of eight terpene derivatives. A new prenylbicyclogermacrene derivative (2) was isolated along with seven other compounds (1, 3-8). Among them, the triterpene ß-boswellic acid (5) showed the strongest activity and reduced 90% of pentylenetetrazole (PTZ)-induced seizures at 100 µg/mL. In parallel to B. sacra, a commercial extract of Boswellia serrata was also evaluated and showed moderate bioactivity (45% reduction at 30 µg/mL). The extract of B. serrata was subjected to targeted isolation of other boswellic acid derivatives (9-13), which were evaluated for antiseizure activity in comparison with 5. In the whole series, ß-boswellic acid (5) was the most active (60% reduction at 200 µM), and its potency was also confirmed with its purchased standard (S5). Pure nanoparticles of S5 and a commercially formulated extract of B. serrata were tested in a PTZ-kindling mouse seizure model. This notably revealed that the S5 administration reduced seizures by 50% in this mouse model, which was consistent with its detection and quantification in plasma and brain samples. This study and the preclinical evaluation performed indicate that ß-boswellic acid, common to various species of Boswellia, has some potential as an antiseizure agent.

Metabolite Profiling of Javanese Ginger Zingiber purpureum and Identification of Antiseizure Metabolites via a Low-Cost Open-Source Zebrafish Bioassay-Guided Isolation.

The rhizomes of Zingiber purpureum, "Bangle", were investigated for its antiseizure properties using a streamlined and cost-effective zebrafish screening strategy and a mouse epilepsy assay. Its hexane extract demonstrated strong antiseizure activity in zebrafish epilepsy assay and was, therefore, selected for bioactivity-guided fractionation. Twelve compounds (1-12) were isolated, and two bioactive phenylbutenoids, trans- (11) and cis-banglene (12), reduced up to 70% of pentylenetetrazole (PTZ)-induced seizures. These compounds showed moderate activity against PTZ-induced seizures in a mouse epilepsy assay. To understand the specificity of Z. purpureum active compounds, its chemical profile was compared to that of Z. officinale. Their composition was assessed by differential metabolite profiling visualized by a molecular network, which revealed only vanillin derivatives and terpenoids as common metabolites and gave a comprehensive view of Z. purpureum composition. This study demonstrates the efficacy of a streamlined zebrafish epilepsy assay, which is therefore suitable for routine screening in phytochemistry laboratories.

Seizing the moment: Zebrafish epilepsy models.

Zebrafish are now widely accepted as a valuable animal model for a number of different central nervous system (CNS) diseases. They are suitable both for elucidating the origin of these disorders and the sequence of events culminating in their onset, and for use as a high-throughput in vivo drug screening platform. The availability of powerful and effective techniques for genome manipulation allows the rapid modelling of different genetic epilepsies and of conditions with seizures as a core symptom. With this review, we seek to summarize the current knowledge about existing epilepsy/seizures models in zebrafish (both pharmacological and genetic) and compare them with equivalent rodent and human studies. New findings obtained from the zebrafish models are highlighted. We believe that this comprehensive review will highlight the value of zebrafish as a model for investigating different aspects of epilepsy and will help researchers to use these models to their full extent.

Antiseizure potential of the ancient Greek medicinal plant Helleborus odorus subsp. cyclophyllus and identification of its main active principles.

ETHNOPHARMACOLOGICAL RELEVANCE: Ethnopharmacological data and ancient texts support the use of black hellebore (Helleborus odorus subsp. cyclophyllus, Ranunculaceae) for the management and treatment of epilepsy in ancient Greece. AIM OF THE STUDY: A pharmacological investigation of the root methanolic extract (RME) was conducted using the zebrafish epilepsy model to isolate and identify the compounds responsible for a potential antiseizure activity and to provide evidence of its historical use. In addition, a comprehensive metabolite profiling of this studied species was proposed. MATERIALS AND METHODS: The roots were extracted by solvents of increasing polarity and root decoction (RDE) was also prepared. The extracts were evaluated for antiseizure activity using a larval zebrafish epilepsy model with pentylenetetrazole (PTZ)-induced seizures. The RME exhibited the highest antiseizure activity and was therefore selected for bioactivity-guided fractionation. Isolated compounds were fully characterized by NMR and high-resolution tandem mass spectrometry (HRMS/MS). The UHPLC-HRMS/MS analyses of the RME and RDE were used for dereplication and metabolite profiling. RESULTS: The RME showed 80% inhibition of PTZ-induced locomotor activity (300 µg/ml). This extract was fractionated and resulted in the isolation of a new glucopyranosyl-deoxyribonolactone (1) and a new furostanol saponin derivative (2), as well as of 20-hydroxyecdysone (3), hellebrin (4), a spirostanol glycoside derivative (5) and deglucohellebrin (6). The antiseizure activity of RME was found to be mainly due to the new furostanol saponin (2) and hellebrin (4), which reduced 45% and 60% of PTZ-induced seizures (135 µM, respectively). Besides, the aglycone of hellebrin, hellebrigenin (S34), was also active (45% at 7 µM). To further characterize the chemical composition of both RME and RDE, 30 compounds (A7-33, A35-37) were annotated based on UHPLC-HRMS/MS metabolite profiling. This revealed the presence of additional bufadienolides, furostanols, and evidenced alkaloids. CONCLUSIONS: This study is the first to identify the molecular basis of the ethnopharmacological use of black hellebore for the treatment of epilepsy. This was achieved using a microscale zebrafish epilepsy model to rapidly quantify in vivo antiseizure activity. The UHPLC-HRMS/MS profiling revealed the chemical diversity of the extracts and the presence of numerous bufadienolides, furostanols and ecdysteroids, also present in the decoction.

Zebrafish bioassay-guided isolation of antiseizure compounds from the Cameroonian medicinal plant Cyperus articulatus L.

BACKGROUND: Epilepsy is a chronic neurological disorder affecting more than 50 million people worldwide, of whom 80% live in low- and middle-income countries. Due to the limited availability of antiseizure drugs (ASDs) in these countries, medicinal plants are the first-line treatment for most epilepsy patients. In Cameroon, a decoction of Cyperus articulatus L. rhizomes is traditionally used to treat epilepsy. PURPOSE: The aim of this study was to identify and isolate the active compounds responsible for the antiseizure activity of C. articulatus in order to confirm both its traditional medicinal usage and previous in vivo studies on extracts of this plant in mouse epilepsy models. METHODS: The dried rhizomes of C. articulatus were extracted with solvents of increasing polaritie (hexane, dichloromethane, methanol and water). A traditional decoction and an essential oil were also prepared. These extracts were evaluated for antiseizure activity using a larval zebrafish seizure model with seizures induced by the GABAA antagonist pentylenetetrazole (PTZ). The hexane extract demonstrated the highest antiseizure activity and was therefore selected for bioassay-guided fractionation. The isolated bioactive compounds were characterized by classical spectroscopic methods. Since they were found to be volatile, they were quantified by GC-FID. In addition, the absorption of the active compounds through the gastrointestinal tract and the blood-brain barrier was evaluated using a hexadecane and a blood-brain barrier parallel artificial membrane permeability assays (HDM-PAMPA and PAMPA-BBB). RESULTS: The hexane extract of C. articulatus exhibited the highest antiseizure activity with a reduction of 93% of PTZ-induced seizures, and was therefore subjected to bioassay-guided fractionation in order to isolate the active principles. Four sesquiterpenoids were identified as cyperotundone (1), mustakone (2), 1,2-dehydro-α-cyperone (3) and sesquichamaenol (4) and exhibited significant antiseizure activity. These volatile compounds were quantified by GC in the hexane extract, the essential oil and the simulated traditional decoction. In addition, the constituents of the hexane extract including compounds 1 and 2 were found to cross the gastrointestinal barrier and the major compound 2 crossed the blood-brain barrier as well. CONCLUSION: These results highlight the antiseizure activity of various sesquiterpene compounds from a hexane extract of C. articulatus dried rhizomes and support its use as a traditional treatment for epilepsy.

l-Isoaspartyl Methyltransferase Deficiency in Zebrafish Leads to Impaired Calcium Signaling in the Brain.

Isomerization of l-aspartyl and l-asparaginyl residues to l-isoaspartyl residues is one type of protein damage that can occur under physiological conditions and leads to conformational changes, loss of function, and enhanced protein degradation. Protein l-isoaspartyl methyltransferase (PCMT) is a repair enzyme whose action initiates the reconversion of abnormal l-isoaspartyl residues to normal l-aspartyl residues in proteins. Many lines of evidence support a crucial role for PCMT in the brain, but the mechanisms involved remain poorly understood. Here, we investigated PCMT activity and function in zebrafish, a vertebrate model that is particularly well-suited to analyze brain function using a variety of techniques. We characterized the expression products of the zebrafish PCMT homologous genes pcmt and pcmtl. Both zebrafish proteins showed a robust l-isoaspartyl methyltransferase activity and highest mRNA transcript levels were found in brain and testes. Zebrafish morphant larvae with a knockdown in both the pcmt and pcmtl genes showed pronounced morphological abnormalities, decreased survival, and increased isoaspartyl levels. Interestingly, we identified a profound perturbation of brain calcium homeostasis in these morphants. An abnormal calcium response upon ATP stimulation was also observed in mouse hippocampal HT22 cells knocked out for Pcmt1. This work shows that zebrafish is a promising model to unravel further facets of PCMT function and demonstrates, for the first time in vivo, that PCMT plays a pivotal role in the regulation of calcium fluxes.

Fishing for a deeper understanding of nicotine effects using zebrafish behavioural models.

Nicotine, the primary psychoactive component of tobacco, is the most widely used drug of abuse. Although the substance is well-known, there is still a lack of information concerning its long-term neurological and physiological effects and its mechanisms of action. In order to search for new, effective drugs in the therapy of nicotinism, as well as to design new drugs that exert positive nicotine-like effects, further experiments are needed, ideally also using new behavioural models and paradigms. A wide range of complex behaviours - including aggression, anxiety, long- and short-term memory, object discrimination and colour preference - have recently been comprehensively classified and characterized in the zebrafish model. Zebrafish offer an attractive experimental platform, based on a microscale in vivo bioassays, which can be used to investigate psychoactive drugs, their effects on the central nervous system and potential treatments of drug addictions. In this review, we present recent data revealing the potential of the zebrafish model to evaluate the effects and molecular mechanisms of nicotine by taking into consideration its impact on anxiety, learning and memory, addiction and social behaviours.

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.

High-performance counter-current chromatography isolation and initial neuroactivity characterization of furanocoumarin derivatives from Peucedanum alsaticum L (Apiaceae).

BACKGROUND: Medicinal plants are a proven source of drug-like small molecules with activity towards targets relevant for diseases of the central nervous system (CNS). Plant species of the Apiaceae family have to date yielded a number of neuroactive metabolites, such as coumarin derivatives with acetylcholinesterase inhibitory activity or anti-seizure activity. PURPOSE: To accelerate the discovery of neuroactive phytochemicals with potential as CNS drug leads, we sought to rapidly isolate furanocoumarins, primary constituents of the dichloromethane (DCM) extract of the fruits of Peucedanum alsaticum L. (Apiaceae), using high-performance counter-current chromatography (HPCCC) and to evaluate their neuroactivity using both in vitro and in vivo microscale bioassays based on cholinesterase ELISAs and zebrafish epilepsy models. RESEARCH METHODS AND PROCEDURE: In this study the DCM extract was subjected to HPCCC for the efficient separation (60 min) and isolation of furanocoumarins. Isolated compounds were identified with TOF-ESI-MS and NMR techniques and examined as inhibitors of AChE and BChE using ELISA microtiter assays. Anti-seizure properties of the extract and of the isolated compounds were evaluated using a zebrafish epilepsy model based on the GABAA antagonist pentylenetetrazol (PTZ), which induces increased locomotor activity and seizure-like behavior. RESULTS: The solvent system, composed of n-heptane, ethyl acetate, methanol and water (3:1:3:1, v/v/v/v), enabled the isolation of 2.63 mg lucidafuranocoumarin A (purity 98%) and 8.82 mg bergamottin (purity 96%) from 1.6 g crude DCM extract. The crude extract, at a concentration of 100 µg/ml, exhibited a weak inhibitory activity against acetylcholinesterase (AChE) (9.63 ±â€¯1.59%) and a moderate inhibitory activity against butyrylcholinestrase (BChE) (49.41 ±â€¯2.19%). Lucidafuranocoumarin A (100 µg/ml) was inactive against AChE but showed moderate inhibition towards BChE (40.66 ±â€¯1.25%). The DCM extract of P. alsaticum fruits (0.62-1.75 µg/ml) and bergamottin (2-10 µm) exhibited weak anti-seizure activity, while lucidafuranocoumarin A (10-16 µm) was found to significantly inhibit PTZ-induced seizures. The percentage of seizure inhibition for the isolated compounds, at their most bioactive concentration, was 26% for bergamottin and 69% for lucidafuranocoumarin A. CONCLUSION: Our findings underscore the utility of HPCCC for the rapid isolation of rare coumarin derivatives, and the potential of microscale in vivo bioassays based on zebrafish disease models for the rapid assessment of neuroactivity of these drug-like natural products.

Deep learning image recognition enables efficient genome editing in zebrafish by automated injections.

One of the most popular techniques in zebrafish research is microinjection. This is a rapid and efficient way to genetically manipulate early developing embryos, and to introduce microbes, chemical compounds, nanoparticles or tracers at larval stages. Here we demonstrate the development of a machine learning software that allows for microinjection at a trained target site in zebrafish eggs at unprecedented speed. The software is based on the open-source deep-learning library Inception v3. In a first step, the software distinguishes wells containing embryos at one-cell stage from wells to be skipped with an accuracy of 93%. A second step was developed to pinpoint the injection site. Deep learning allows to predict this location on average within 42 µm to manually annotated sites. Using a Graphics Processing Unit (GPU), both steps together take less than 100 milliseconds. We first tested our system by injecting a morpholino into the middle of the yolk and found that the automated injection efficiency is as efficient as manual injection (~ 80%). Next, we tested both CRISPR/Cas9 and DNA construct injections into the zygote and obtained a comparable efficiency to that of an experienced experimentalist. Combined with a higher throughput, this results in a higher yield. Hence, the automated injection of CRISPR/Cas9 will allow high-throughput applications to knock out and knock in relevant genes to study their mechanisms or pathways of interest in diverse areas of biomedical research.

Phenotypic assays in yeast and zebrafish reveal drugs that rescue ATP13A2 deficiency.

Mutations in ATP13A2 (PARK9) are causally linked to the rare neurodegenerative disorders Kufor-Rakeb syndrome, hereditary spastic paraplegia and neuronal ceroid lipofuscinosis. This suggests that ATP13A2, a lysosomal cation-transporting ATPase, plays a crucial role in neuronal cells. The heterogeneity of the clinical spectrum of ATP13A2-associated disorders is not yet well understood and currently, these diseases remain without effective treatment. Interestingly, ATP13A2 is widely conserved among eukaryotes, and the yeast model for ATP13A2 deficiency was the first to indicate a role in heavy metal homeostasis, which was later confirmed in human cells. In this study, we show that the deletion of YPK9 (the yeast orthologue of ATP13A2) in Saccharomyces cerevisiae leads to growth impairment in the presence of Zn2+, Mn2+, Co2+ and Ni2+, with the strongest phenotype being observed in the presence of zinc. Using the ypk9Δ mutant, we developed a high-throughput growth rescue screen based on the Zn2+ sensitivity phenotype. Screening of two libraries of Food and Drug Administration-approved drugs identified 11 compounds that rescued growth. Subsequently, we generated a zebrafish model for ATP13A2 deficiency and found that both partial and complete loss of atp13a2 function led to increased sensitivity to Mn2+. Based on this phenotype, we confirmed two of the drugs found in the yeast screen to also exert a rescue effect in zebrafish-N-acetylcysteine, a potent antioxidant, and furaltadone, a nitrofuran antibiotic. This study further supports that combining the high-throughput screening capacity of yeast with rapid in vivo drug testing in zebrafish can represent an efficient drug repurposing strategy in the context of rare inherited disorders involving conserved genes. This work also deepens the understanding of the role of ATP13A2 in heavy metal detoxification and provides a new in vivo model for investigating ATP13A2 deficiency.

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.