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

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

Behavioral profile of zebrafish larvae during a one-hour exposure to vehicle or PTZ.

<p>The average movement (y-axis) of vehicle- and 20 mM PTZ-treated larvae are denoted by closed triangles (grey) and closed circles (black) respectively. The mean movement ± SD of 12 larvae is depicted per minute (x-axis) of the tracking session. By 15 min, the frequency of convulsion-like episodes in all larvae reached a rate of one or more per minute, which is reflected in the decrease in SD after this time point.</p

Electrographic activity in zebrafish optic tecta: fragments of representative recordings.

<p>(A) Vehicle only; (B – P) 20 mM PTZ with the following pre-treatment: (B) VHC; (C) CBZ; (D) DZP; (E) ETS; (F) GBP; (G) LTG; (H) LVT; (I) OXC; (J) PHT; (K) PMD; (L) TGB; (M) TPR; (N) VPA; (O) ZSM; (P) ASP. Recordings were performed in current clamp mode, low-pass filtered at 1 kHz, high-pass filtered 0.1 Hz, digital gain 10, sampling interval 10 µs.</p

Quantitative analysis of interictal-like electrographic activity in response to AEDs.

<p>(A) Total number of interictal-like events within a 10-min recording (mean±SEM); (B) duration of interictal-like events in seconds within a 10-min recording (mean±SEM). Values that were significantly different from PTZ control were determined using one-way ANOVA with *, ** and *** denoting p<0.05, p<0.01 and p<0.001 respectively. The number of recordings analysed were: VHC (n = 8), PTZ (n = 9), CBZ (n = 8), DZP (n = 8), ETS (n = 9), GBP (n = 8), LTG (n = 8), LVT (n = 8), OXC (n = 8), PHT (n = 8), PMD (n = 8), TGB (n = 8), TPR (n = 8), VPA (n = 8), ZSM (n = 8), ASP (n = 8); F values (treatment group-dependent variability): (A) 4.298, (B) 6.602.</p

Behavioral profile of zebrafish larvae to AEDs.

<p>(A) The top graph depicts the average larval locomotor activity within 30 minutes (y-axis) relative to VHC+PTZ (PTZ) control, as depicted by the average % ± SEM; treatment groups where average movement was significantly decreased compared to PTZ (one-way ANOVA) are marked *, **, and *** (p<0.05, p<0.01, and p<0.001, respectively). (B-O) The average total movement (y-axis) of larvae treated with VHC only or PTZ with either VHC or AEDs. The average larval movement is depicted per 5 min interval (x-axis) of the tracking session. Time points when the average movement was significantly decreased compared to PTZ control (repeated measures ANOVA) are marked *, **, and *** (p<0.05, p<0.01, and p<0.001, respectively); standard errors are shown. F values (treatment group-dependent variability): (A) 55.88, (B) 77.26, (C) 144.3, (D) 107.6, (E) 124.7, (F) 65.73, (G) 80.58, (H) 79.56, (I) 70.39, (J) 153.2, (K) 70.29, (L) 80.36, (M) 97.76, (N) 68.3, (O) 137.8. Total number of larvae used are indicated in brackets.</p

Quantitative analysis of ictal-like electrographic activity in response to AEDs.

<p>(A) Number of ictal-like events within a 10-min recording (mean±SEM); (B) duration of ictal-like events within a 10-min recording (mean±SEM); (C) total cumulative duration of all types of epileptiform activity measured. Values that were significantly different from PTZ control were determined using the one-way ANOVA with *, ** and *** denoting p<0.05, p<0.01 and p<0.001 respectively. The number of recordings analysed were: VHC (n = 8), PTZ (n = 9), CBZ (n = 8), DZP (n = 8), ETS (n = 9), GBP (n = 8), LTG (n = 8), LVT (n = 8), OXC (n = 8), PHT (n = 8), PMD (n = 8), TGB (n = 8), TPR (n = 8), VPA (n = 8), ZSM (n = 8), ASP (n = 8). F values (treatment group-dependent variability): (A) 13.45, (B) 3.056, (C) 11.41.</p

Comparison of AED activity in the zebrafish and rodent acute PTZ models.

<p>An AED is indicated as positive for 30-min integration intervals if it significantly (p<0.05) decreased locomotor activity. We considered an AED positive (+) in the 5-min time slices if there were one or more points significantly different from the PTZ-treated group (p<0.01 or better). Slight activity (+/−) was indicated when only one time point was significantly lower (p<0.05) than the corresponding control point. ^a, spike-wave discharges (SWD) can not be directly measured in zebrafish tectal field recordings; NA, data not available.</p

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.status: publishe