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Large-Scale Phenotype-Based Antiepileptic Drug Screening in a Zebrafish Model of Dravet Syndrome(1,2,3).

Dinday MT, Baraban SC - eNeuro (2015)

Bottom Line: Mutations in a voltage-gated sodium channel (SCN1A) result in Dravet Syndrome (DS), a catastrophic childhood epilepsy.The effectiveness of compounds that block neuronal calcium current (dimethadione) or enhance serotonin signaling (fenfluramine) in our zebrafish model suggests that these may be important therapeutic targets in patients with DS.Over 150 compounds resulting in fatality were also identified.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurological Surgery, Epilepsy Research Laboratory, University of California San Francisco , San Francisco, California 94143.

ABSTRACT
Mutations in a voltage-gated sodium channel (SCN1A) result in Dravet Syndrome (DS), a catastrophic childhood epilepsy. Zebrafish with a mutation in scn1Lab recapitulate salient phenotypes associated with DS, including seizures, early fatality, and resistance to antiepileptic drugs. To discover new drug candidates for the treatment of DS, we screened a chemical library of ∼1000 compounds and identified 4 compounds that rescued the behavioral seizure component, including 1 compound (dimethadione) that suppressed associated electrographic seizure activity. Fenfluramine, but not huperzine A, also showed antiepileptic activity in our zebrafish assays. The effectiveness of compounds that block neuronal calcium current (dimethadione) or enhance serotonin signaling (fenfluramine) in our zebrafish model suggests that these may be important therapeutic targets in patients with DS. Over 150 compounds resulting in fatality were also identified. We conclude that the combination of behavioral and electrophysiological assays provide a convenient, sensitive, and rapid basis for phenotype-based drug screening in zebrafish mimicking a genetic form of epilepsy.

No MeSH data available.


Related in: MedlinePlus

Positive hits identified in the locomotion assay. a, Heat map showing the results of individual zebrafish trials (1-6) for compounds tested at a concentration of 100 µm in the locomotion-tracking assay. Raw data values for individual fish are shown within the color-coded boxes for one sample trial. Mean velocity data are shown at right for “trial 1” and “trial 2”; six fish per trial. Note: only drugs highlighted in bold type were classified as positive nontoxic hits in two independent trials and moved on to further testing. b, Representative raw locomotion data plots for six individual scn1Lab mutant larvae at baseline (top) and following the addition of a compound resulting in fatality (bottom, gemfibrozil) or hyperactivity (bottom, mepivacaine). Movement is color coded, with low-velocity movements shown in yellow, and high velocity movements shown in red.
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Figure 2: Positive hits identified in the locomotion assay. a, Heat map showing the results of individual zebrafish trials (1-6) for compounds tested at a concentration of 100 µm in the locomotion-tracking assay. Raw data values for individual fish are shown within the color-coded boxes for one sample trial. Mean velocity data are shown at right for “trial 1” and “trial 2”; six fish per trial. Note: only drugs highlighted in bold type were classified as positive nontoxic hits in two independent trials and moved on to further testing. b, Representative raw locomotion data plots for six individual scn1Lab mutant larvae at baseline (top) and following the addition of a compound resulting in fatality (bottom, gemfibrozil) or hyperactivity (bottom, mepivacaine). Movement is color coded, with low-velocity movements shown in yellow, and high velocity movements shown in red.

Mentions: Locomotion tracking is a reliable and rapid strategy with which to monitor behavioral seizures in freely swimming larval zebrafish (Baraban et al., 2005, 2013; Winter et al., 2008). In these locomotion plots, high-velocity movements of ≥20 mm/s correspond to paroxysmal whole-body convulsions, referred to as Stage III, and are consistently observed in unprovoked scn1Lab mutant larvae but not in age-matched wild-type siblings. Using automated locomotion tracking, we performed a phenotype-based screen to identify compounds that significantly reduce mutant swim behavior to levels associated with Stage 0 or Stage I (e.g., activity equivalent to that seen in normal untreated WT zebrafish). In a 96-well format, we tracked mutant swim activity at baseline, and then again after addition of a test compound (100 µm); each compound was tested on six individual mutant larvae (Fig. 1a), and larvae were sorted based on pigmentation differences (Fig. 1b). Mutant swim activity between two consecutive recording epochs in embryo media is tracked on every plate as an internal control. A box plot showing the change in swim velocity in untreated mutants is shown in Figure 1c (n = 112) and defined as the control. Based on an SD of 21.8 for these data, we set the detection threshold as any compound that inhibits movement (measured as a change in mean velocity) by >2 SDs (or ≥44%). This approach was previously validated using standard antiepileptic drugs in this model (Baraban et al., 2013). Next, we screened a repurposed library in which all compounds have reached the clinical evaluation stage (PHARMAKON 1600 Collection; http://www.msdiscovery.com/pharma.html). Among the 1012 compounds screened (Fig. 1d) only 20 (or 1.97%) were found to significantly inhibit spontaneous seizure behavior in scn1Lab mutants. All 20 compounds were subsequently retested in a separate clutch of scn1Lab mutants at a concentration of 100 µm (Fig. 2a, trial 2; N = 6 fish/compound). A total of 154 compounds were classified as “toxic” (Table 2); 55 compounds were classified as “hyperexcitable” (Table 3). Representative locomotion tracking raw data plots for gemfibrozil, a toxic nonsteroid nuclear receptor ligand, and mepivacaine, a hyperexcitable proconvulsant anesthetic, are shown in Figure 2b.


Large-Scale Phenotype-Based Antiepileptic Drug Screening in a Zebrafish Model of Dravet Syndrome(1,2,3).

Dinday MT, Baraban SC - eNeuro (2015)

Positive hits identified in the locomotion assay. a, Heat map showing the results of individual zebrafish trials (1-6) for compounds tested at a concentration of 100 µm in the locomotion-tracking assay. Raw data values for individual fish are shown within the color-coded boxes for one sample trial. Mean velocity data are shown at right for “trial 1” and “trial 2”; six fish per trial. Note: only drugs highlighted in bold type were classified as positive nontoxic hits in two independent trials and moved on to further testing. b, Representative raw locomotion data plots for six individual scn1Lab mutant larvae at baseline (top) and following the addition of a compound resulting in fatality (bottom, gemfibrozil) or hyperactivity (bottom, mepivacaine). Movement is color coded, with low-velocity movements shown in yellow, and high velocity movements shown in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4596025&req=5

Figure 2: Positive hits identified in the locomotion assay. a, Heat map showing the results of individual zebrafish trials (1-6) for compounds tested at a concentration of 100 µm in the locomotion-tracking assay. Raw data values for individual fish are shown within the color-coded boxes for one sample trial. Mean velocity data are shown at right for “trial 1” and “trial 2”; six fish per trial. Note: only drugs highlighted in bold type were classified as positive nontoxic hits in two independent trials and moved on to further testing. b, Representative raw locomotion data plots for six individual scn1Lab mutant larvae at baseline (top) and following the addition of a compound resulting in fatality (bottom, gemfibrozil) or hyperactivity (bottom, mepivacaine). Movement is color coded, with low-velocity movements shown in yellow, and high velocity movements shown in red.
Mentions: Locomotion tracking is a reliable and rapid strategy with which to monitor behavioral seizures in freely swimming larval zebrafish (Baraban et al., 2005, 2013; Winter et al., 2008). In these locomotion plots, high-velocity movements of ≥20 mm/s correspond to paroxysmal whole-body convulsions, referred to as Stage III, and are consistently observed in unprovoked scn1Lab mutant larvae but not in age-matched wild-type siblings. Using automated locomotion tracking, we performed a phenotype-based screen to identify compounds that significantly reduce mutant swim behavior to levels associated with Stage 0 or Stage I (e.g., activity equivalent to that seen in normal untreated WT zebrafish). In a 96-well format, we tracked mutant swim activity at baseline, and then again after addition of a test compound (100 µm); each compound was tested on six individual mutant larvae (Fig. 1a), and larvae were sorted based on pigmentation differences (Fig. 1b). Mutant swim activity between two consecutive recording epochs in embryo media is tracked on every plate as an internal control. A box plot showing the change in swim velocity in untreated mutants is shown in Figure 1c (n = 112) and defined as the control. Based on an SD of 21.8 for these data, we set the detection threshold as any compound that inhibits movement (measured as a change in mean velocity) by >2 SDs (or ≥44%). This approach was previously validated using standard antiepileptic drugs in this model (Baraban et al., 2013). Next, we screened a repurposed library in which all compounds have reached the clinical evaluation stage (PHARMAKON 1600 Collection; http://www.msdiscovery.com/pharma.html). Among the 1012 compounds screened (Fig. 1d) only 20 (or 1.97%) were found to significantly inhibit spontaneous seizure behavior in scn1Lab mutants. All 20 compounds were subsequently retested in a separate clutch of scn1Lab mutants at a concentration of 100 µm (Fig. 2a, trial 2; N = 6 fish/compound). A total of 154 compounds were classified as “toxic” (Table 2); 55 compounds were classified as “hyperexcitable” (Table 3). Representative locomotion tracking raw data plots for gemfibrozil, a toxic nonsteroid nuclear receptor ligand, and mepivacaine, a hyperexcitable proconvulsant anesthetic, are shown in Figure 2b.

Bottom Line: Mutations in a voltage-gated sodium channel (SCN1A) result in Dravet Syndrome (DS), a catastrophic childhood epilepsy.The effectiveness of compounds that block neuronal calcium current (dimethadione) or enhance serotonin signaling (fenfluramine) in our zebrafish model suggests that these may be important therapeutic targets in patients with DS.Over 150 compounds resulting in fatality were also identified.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurological Surgery, Epilepsy Research Laboratory, University of California San Francisco , San Francisco, California 94143.

ABSTRACT
Mutations in a voltage-gated sodium channel (SCN1A) result in Dravet Syndrome (DS), a catastrophic childhood epilepsy. Zebrafish with a mutation in scn1Lab recapitulate salient phenotypes associated with DS, including seizures, early fatality, and resistance to antiepileptic drugs. To discover new drug candidates for the treatment of DS, we screened a chemical library of ∼1000 compounds and identified 4 compounds that rescued the behavioral seizure component, including 1 compound (dimethadione) that suppressed associated electrographic seizure activity. Fenfluramine, but not huperzine A, also showed antiepileptic activity in our zebrafish assays. The effectiveness of compounds that block neuronal calcium current (dimethadione) or enhance serotonin signaling (fenfluramine) in our zebrafish model suggests that these may be important therapeutic targets in patients with DS. Over 150 compounds resulting in fatality were also identified. We conclude that the combination of behavioral and electrophysiological assays provide a convenient, sensitive, and rapid basis for phenotype-based drug screening in zebrafish mimicking a genetic form of epilepsy.

No MeSH data available.


Related in: MedlinePlus