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

Development and validation of zebrafish seizure models for drug discovery

Tatiana Afrikanova 14.00 Validation of an animal model constitutes a large partof successful drug development. In this thesis we describe the development andvalidation of in vivo models of seizures in zebrafish larvae. The process of building an animal model forneurological disorders consists of several steps of development and systematicevaluation of their quality and relevance. Based on a hypothesis aboutbrain-behavior relationships, a model is developed and tested. Validation of a model is a scientific method toimprove the confidence in a model. Validity is defined as the agreement betweena test score and the quality it is believed to measure. Validity is restrictedto a specific use of a model, and must always be open for discussion andre-evaluation. Most animal models are evaluated based on the criteria proposedby Willner: construct, predictive and face validity. Other important factorsare animal welfare, reliability and replicability as well as cross-speciessimilarity. Epilepsy is one of the most common neurologicaldisorders with a high disabling risk; the main symptom of epilepsy is theoccurrence of seizures. An epileptic seizure (according to ILAE) is a transientoccurrence of signs and/or symptoms due to abnormal excessive or synchronousneuronal activity in the brain. Epilepsy is defined as a disorder of the braincharacterized by en enduring predisposition to generate epileptic seizures andby the neurobiologic, cognitive, physiological, and social consequences of thiscondition. There are cases reported where seizures stop occurringspontaneously (self-resolving). Still, in the majority of patients, adequateseizure control or even seizure freedom is achieved with the help ofantiepileptic drugs (AEDs). To date, there are several generations of AEDs onthe market with different or multiple mechanisms of action available,nevertheless, about one third of people diagnosed with epilepsy do not respondto the prescribed treatment. Some of the non-responders can be cured bysurgical intervention removing the brain area involved in seizure generation.Other alternative treatments include ketogenic diet and vagus nervestimulation. To start with, epilepsy involves excessive and/orhighly synchronized neuronal firing, which is otherwise a normal function ofthe brain. Therefore, due to the complexity of disease it s not possible tomodel it in a biochemical in vitro assay. The use of the existing -predominantly rodent -models of seizures and epilepsy is limited due to theassay costs, timing and labour-intensity despite the value of the data theyprovide. The constant trade of validity against resourceconsumption leads us to a different approach in model building. It is possibleto use smaller vertebrates to increase the assay s throughput. On the otherhand, the decrease in construct validity in this case can be partially compensated by the convenience of geneticmanipulation, visual transparency and automation solutions. Assuming that asimpler organism feels less pain, ethical considerations also support zebrafishresearch, as the zebrafish larvae are not even considered laboratory animalsbefore day 6 of life. Zebrafish video tracking haverecently emerged as an attractive in vivo model for epilepsy.Seven-day-old zebrafish larvae exposed to the GABAA antagonistpentylenetetrazol (PTZ) exhibit increased locomotor activity, seizure-likebehavior, and epileptiform electrographic activity. A previous study showedthat 12 out of 13 AEDs suppressed PTZ-mediated increases in larval movement,indicating the potential utility of zebrafish as a high-throughput in vivo modelfor AED discovery. However, a question remained as to whether an AED-induceddecrease in locomotion is truly indicative of anticonvulsant activity, as somedrugs may impair larval movement through other mechanisms such as generaltoxicity or sedation. We therefore carried out a study in PTZ-treated zebrafishlarvae, to directly compare the ability of AEDs to inhibit seizure-likebehavioral manifestations with their capacity to suppress epileptiformelectrographic activity. We re-tested the 13 AEDs of which 12 were previouslyreported to inhibit convulsions in the larval movement tracking assay, administeringconcentrations that did not, on their own, impair locomotion. In parallel, wequantified the parameters of open-field recordings on larval brains aftertreatment with each AED. For 10 of AEDs tested we obtained the same response inboth the behavioral and electrographic assays. Overall our data correlate wellwith those reported in the literature for acute rodent PTZ tests, indicatingthat the larval zebrafish brain is more discriminatory than previously thoughtin its response to AEDs with different modes of action. Our results underscorethe validity of using the zebrafish larval locomotor assay as a rapidfirst-pass screening tool in assessing the anticonvulsant and/or proconvulsantactivity of compounds, but also highlight the importance of performing adequatevalidation when using in vivo models. The other question explored was ifzebrafish could be used as a model organism for treatment-resistant seizures.Refractory seizures affect about a third of patients suffering from epilepsy.To fulfill the need for new medications targeting these difficult-to-treatcases, chronic rodent models offer the opportunity to study mechanisms of drugresistance. The use of such models, however, has been rather limited, as allhave proven to be time-consuming and labor-intensive. In this study, wecharacterize the allylglycine (AG) seizure model, a simple in vivo modelshowing a high level of treatment resistance. (D,L)-Allylglycine inhibitsglutamic acid decarboxylase the key enzyme in GABA biosynthesis leading toGABA depletion, seizures and neuronal damage. We performed a side-by-sidecomparison of mouse and zebrafish acute AG treatment including biochemical,electrographic and behavioral assessment. Interestingly, seizure progressionrate and GABA depletion kinetics were comparable in both species. Fivemechanistically diverse AEDs (valproate, diazepam, levetiracetam, phenytoin andtopiramate) were used. All tested AEDs showed a limited protective effect(mainly mortality delay) at doses close to TD50 (doseinducing motor impairment in 50% of animals) in mice; experiments in fishdemonstrated a complete protective effect ofVPA, while other AEDs were only partially or not active in the assays run.Having demonstrated cross-species similarities, we conclude that the use of AGin zebrafish could allow for high-throughput drug screening and thereforepropose it as a convenient model in the context of treatment-resistantseizures. Normal 0 21 false false false EN-US X-NONE X-NONE <w:LsdException Locked="false" Priority="19" SemiHidden="false" UnhideWhenUsed="false" QFormat="true" Name="Subtle Em

Analysis of antibiotic resistance markers in Chlamydia trachomatis clinical isolates obtained after ineffective antibiotic therapy

Antibiotic resistance markers were analyzed in C. trachomatis clinical isolates obtained after ineffective therapy of urogenital chlamydiasis with fluoroquinolones and macrolides. Heterotypical resistance to fluoroquinolones and macrolides was detected in all clinical isolates. No significant mutations in the target genes were detected.status: publishe

Insights from Zebrafish and Mouse Models on the Activity and Safety of Ar-Turmerone as a Potential Drug Candidate for the Treatment of Epilepsy

In a previous study, we uncovered the anticonvulsant properties of turmeric oil and its sesquiterpenoids (ar-turmerone, α-, β-turmerone and α-atlantone) in both zebrafish and mouse models of chemically-induced seizures using pentylenetetrazole (PTZ). In this follow-up study, we aimed at evaluating the anticonvulsant activity of ar-turmerone further. A more in-depth anticonvulsant evaluation of ar-turmerone was therefore carried out in the i.v. PTZ and 6-Hz mouse models. The potential toxic effects of ar-turmerone were evaluated using the beam walking test to assess mouse motor function and balance. In addition, determination of the concentration-time profile of ar-turmerone was carried out for a more extended evaluation of its bioavailability in the mouse brain. Ar-turmerone displayed anticonvulsant properties in both acute seizure models in mice and modulated the expression patterns of two seizure-related genes (c-fos and brain-derived neurotrophic factor [bdnf]) in zebrafish. Importantly, no effects on motor function and balance were observed in mice after treatment with ar-turmerone even after administering a dose 500-fold higher than the effective dose in the 6-Hz model. In addition, quantification of its concentration in mouse brains revealed rapid absorption after i.p. administration, capacity to cross the BBB and long-term brain residence. Hence, our results provide additional information on the anticonvulsant properties of ar-turmerone and support further evaluation towards elucidating its mechanism of action, bioavailability, toxicity and potential clinical application.status: publishe

Validation of the Zebrafish Pentylenetetrazol Seizure Model: Locomotor versus Electrographic Responses to Antiepileptic Drugs

Zebrafish have recently emerged as an attractive in vivo model for epilepsy. Seven-day-old zebrafish larvae exposed to the GABAA antagonist pentylenetetrazol (PTZ) exhibit increased locomotor activity, seizure-like behavior, and epileptiform electrographic activity. A previous study showed that 12 out of 13 antiepileptic drugs (AEDs) suppressed PTZ-mediated increases in larval movement, indicating the potential utility of zebrafish as a high-throughput in vivo model for AED discovery. However, a question remained as to whether an AED-induced decrease in locomotion is truly indicative of anticonvulsant activity, as some drugs may impair larval movement through other mechanisms such as general toxicity or sedation. We therefore carried out a study in PTZ-treated zebrafish larvae, to directly compare the ability of AEDs to inhibit seizure-like behavioral manifestations with their capacity to suppress epileptiform electrographic activity. We re-tested the 13 AEDs of which 12 were previously reported to inhibit convulsions in the larval movement tracking assay, administering concentrations that did not, on their own, impair locomotion. In parallel, we carried out open-field recordings on larval brains after treatment with each AED. For the majority of AEDs we obtained the same response in both the behavioral and electrographic assays. Overall our data correlate well with those reported in the literature for acute rodent PTZ tests, indicating that the larval zebrafish brain is more discriminatory than previously thought in its response to AEDs with different modes of action. Our results underscore the validity of using the zebrafish larval locomotor assay as a rapid firstpass screening tool in assessing the anticonvulsant and/or proconvulsant activity of compounds, but also highlight the importance of performing adequate validation when using in vivo models.status: publishe

Validation of the zebrafish pentylenetetrazol seizure model: locomotor versus electrographic responses to antiepileptic drugs.

Zebrafish have recently emerged as an attractive in vivo model for epilepsy. Seven-day-old zebrafish larvae exposed to the GABA(A) antagonist pentylenetetrazol (PTZ) exhibit increased locomotor activity, seizure-like behavior, and epileptiform electrographic activity. A previous study showed that 12 out of 13 antiepileptic drugs (AEDs) suppressed PTZ-mediated increases in larval movement, indicating the potential utility of zebrafish as a high-throughput in vivo model for AED discovery. However, a question remained as to whether an AED-induced decrease in locomotion is truly indicative of anticonvulsant activity, as some drugs may impair larval movement through other mechanisms such as general toxicity or sedation. We therefore carried out a study in PTZ-treated zebrafish larvae, to directly compare the ability of AEDs to inhibit seizure-like behavioral manifestations with their capacity to suppress epileptiform electrographic activity. We re-tested the 13 AEDs of which 12 were previously reported to inhibit convulsions in the larval movement tracking assay, administering concentrations that did not, on their own, impair locomotion. In parallel, we carried out open-field recordings on larval brains after treatment with each AED. For the majority of AEDs we obtained the same response in both the behavioral and electrographic assays. Overall our data correlate well with those reported in the literature for acute rodent PTZ tests, indicating that the larval zebrafish brain is more discriminatory than previously thought in its response to AEDs with different modes of action. Our results underscore the validity of using the zebrafish larval locomotor assay as a rapid first-pass screening tool in assessing the anticonvulsant and/or proconvulsant activity of compounds, but also highlight the importance of performing adequate validation when using in vivo models

Evaluation of the anticonvulsant activity of ar-turmerone (AT) as determined by the 6-Hz model.

<p>A) After 30 min of i.p. administration, complete protection was observed for ar-turmerone at a dose range of 0.1 - 50 mg/kg. A group of vehicle-treated mice (VHC) was included as a negative control. Sodium valproate at 300 mg/kg (VAL300) and levetiracetam at 50 mg/kg (LEV50) were used as positive controls. B) After 24 h i.p. administration (50 mg/kg), protection was observed after electrical induction. Statistically significant differences between control (VHC) and sample groups are labeled as * for p < 0.05 (Fisher’s exact test).</p

Representative HPLC chromatograms of mouse brain homogenate analysis for ar-turmerone determination.

<p>A) blank mouse brain extract, B) blank mouse brain extract spiked with ar-turmerone (AT) and internal standard (IS) and C) brain extract sample of a dosed mouse. </p

Evaluation of the effect of ar-turmerone on mouse motor function and balance.

<p>An intravenous dose of 50 mg/kg of ar-turmerone (AT) does not cause any alteration of motor skills in mice. Sensitivity of this model was confirmed by detection of motor and balance deficits induced by diazepam (DPZ) in mice at 1 mg/kg. Compared to control group (VHC), DZP-treated mice displayed a significant increase in number of footslips (A), falls (B) and time to reach goal box (C). Statistically significant differences between control and sample groups are labeled as ** for p < 0.001 (one-way ANOVA test).</p

Anticonvulsant activity of bisabolene sesquiterpenoids of Curcuma longa in zebrafish and mouse seizure models.

urmeric, obtained from the rhizomes of Curcuma longa, is used in South Asia as a traditional medicine for the treatment of epilepsy. To date, in vivo studies on the anticonvulsant activity of turmeric have focused on its principal curcuminoid, curcumin. However, poor absorption and rapid metabolism have limited the therapeutic application of curcumin in humans. To explore the therapeutic potential of turmeric for epilepsy further, we analyzed its anticonvulsant activity in a larval zebrafish seizure assay. Initial experiments revealed that the anticonvulsant activity of turmeric in zebrafish larvae cannot be explained solely by the effects of curcumin. Zebrafish bioassay-guided fractionation of turmeric identified bisabolene sesquiterpenoids as additional anticonvulsants that inhibit PTZ-induced seizures in both zebrafish and mice. Here, we present the first report of the anticonvulsant properties of bisabolene sesquiterpenoids and provide evidence which warrants further investigation toward the mechanistic understanding of their neuromodulatory activity