Seizure suppression by gain-of-function escargot mutations

Abstract

Suppressor mutations provide potentially powerful tools for examining mechanisms underlying neurological disorders and identifying novel targets for pharmacological intervention. Here we describe mutations that suppress seizures in a Drosophila model of human epilepsy. A screen utilizing the Drosophila easily shocked (eas) "epilepsy" mutant identified dominant suppressors of seizure sensitivity. Among several mutations identified, neuronal escargot (esg) reduced eas seizures almost 90%. The esg gene encodes a member of the snail family of transcription factors. Whereas esg is normally expressed in a limited number of neurons during a defined period of nervous system development, here normal esg was expressed in all neurons and throughout development. This greatly ameliorated both the electrophysiological and the behavioral epilepsy phenotypes of eas. Neuronal esg appears to act as a general seizure suppressor in the Drosophila epilepsy model as it reduces the susceptibility of several seizure-prone mutants. We observed that esg must be ectopically expressed during nervous system development to reduce seizure susceptibility in adults. Furthermore, induction of esg in a small subset of neurons (interneurons) will reduce seizure susceptibility. A combination of microarray and computational analyses revealed 100 genes that represent possible targets of neuronal esg. We anticipate that some of these genes may ultimately serve as targets for novel antiepileptic drugs.

Figure 1.—

Screen for eas suppressors reveals multiple escargot alleles.The three strongest gain-of-function eas suppressors identified in our screen (EP684, EP2009, and EP633) all carry EP insertions in the same gene, esg. All three of these esg^EP insertions (solid triangles) occur in the 5′-flanking region of the esg gene. Two additional esg^EP insertions (shaded triangles) also show some suppression of bang sensitivity. One of these (EP2408) occurs in the 5′-flanking region of the esg gene, whereas the other (EP2159) is in the 5′-noncoding region of the esg transcript. A sixth esg^EP insertion (EP683, open triangle) does not show any suppression of bang sensitivity. The EP element in esg^EP683 is located in the 5′-flanking region of esg but is inserted in the opposite orientation of the other five esg^EP insertions; GAL4 induction of esg^EP683 should not induce the level of esg transcript.The escargot ORF (large solid box) encodes a transcription factor with five Zn²⁺-finger DNA-binding domains (open stripes). The escargot protein also possesses a P-DLS-K domain (shaded stripe), which probably binds the dCtBP corepressor.

Figure 2.—

Neuronal expression of esg^EP suppresses a variety of bang-sensitive mutants. Flies of different genotypes were examined for the bang-sensitive behavioral phenotype in response to a 10-sec mechanical bang. The data reveal that elav-GAL4-driven esg^EP acts as a general suppressor of bang-sensitive paralysis: several esg^EP alleles act as suppressors, and different bang-sensitive mutations are suppressed. (A) Ectopic expression of different esg^EP alleles suppresses the bang-sensitive behavioral phenotype of the eas mutation. In the case of esg^EP684 (EP684, solid bar, genotype eas; esg^EP684/+; elav-GAL4/+), ectopic expression driven by elav-GAL4 produces considerable suppression of the eas phenotype: only 4% of flies show paralysis compared with 100% of their sibling controls (open bar, genotype eas; +/CyO; elav-GAL4/+). Substantial suppression is also seen for the esg^EP alleles EP2009 and EP633 (11 and 20% bang sensitivity, respectively). For all genotypes, n > 135. (B) Ectopic expression of different esg^EP alleles also suppresses the bang-sensitive behavioral phenotype of the sda mutation. In the case of esg^EP684 (EP684, solid bar, genotype esg^EP684/+; elav-GAL4 sda/sda), ectopic expression driven by elav-GAL4 produces marked suppression of the sda phenotype: only 2% are bang sensitive compared with 100% of the sibling control flies (open bars, genotype +/CyO; elav-GAL4 sda/sda). Substantial suppression is also seen for the esg^EP alleles EP2009 and EP633 (4 and 11% bang sensitivity, respectively). For all genotypes, n > 135. (C) The behavioral phenotype of the bss/+ semidominant genotype is suppressed by neuronal expression of different esg^EP alleles. For bss/+ sibling control flies (open bars, genotype bss/+; +/CyO; elav-GAL4/+) >50% displayed bang sensitivity. Note that this behavioral response differs somewhat from others described in this article in that flies demonstrate paralysis, but not hyperactivity. This has sometimes been referred to as “stress sensitivity” (Homyk 1977). In contrast, 0% bang-sensitive paralysis is observed in bss/+ siblings carrying esg^EP684 (solid bars, genotype bss/+; esg^EP684/+; elav-GAL4/+). Substantial suppression is also seen for the esg^EP alleles EP2009 and EP633 (5 and 10% bang sensitivity, respectively). For all genotypes, n > 95.

Figure 3.—

Neuronal expression esg^EP raises the seizure threshold of eas flies. (A) A seizure is elicited in an eas sibling control fly (genotype eas; +/CyO; elav-GAL4/+) by a high-frequency stimulus of low strength (8 V). The HF stimulus (box) is a short wave train (0.5-ms pulses at 200 Hz for 300 msec) of electrical stimuli delivered to the brain. The recording is from a DLM and reflects the abnormal HF firing of the innervating DLM motoneuron. The vertical calibration bar is 20 mV and the horizontal bar is 200 msec. (B) A low-voltage HF stimulus (8 V) fails to elicit a seizure in an eas fly carrying the esg^EP2009 suppressor (genotype eas; esg^EP2009/+; elav-GAL4/+) because the stimulus is below the intensity required for a higher seizure threshold. (C) A higher-voltage HF stimulus (20 V) elicits a seizure in an eas fly carrying the esg^EP2009 suppressor (genotype eas; esg^EP2009/+; elav-GAL4/+) because the stimulus is of sufficient intensity to exceed the higher seizure threshold of this fly. (D) HF stimuli of varying voltages were delivered to eas control flies or their siblings that were eas and carried the esg^EP2009 suppressor to determine their seizure thresholds. The threshold curves show the percentage of flies that have seizures following an HF stimulus of a particular voltage. Fifteen eas control flies and 20 of their esg^EP2009-carrying siblings were tested. Note that the esg^EP2009 curve is broader than the control one, indicating a greater range of threshold values. (E) The esg^EP suppressors raise the average seizure threshold for eas by approximately twofold. The mean seizure threshold of eas; +/CyO; elav-GAL4/+ control flies is 7.6 ± 1.9 V. The mean seizure threshold of eas flies carrying any one of the three esg^EP alleles (genotype eas; esg/+; elav-GAL4/+) is higher (EP684, 15.5 ± 5.7 V; EP2009, 15.5 ± 6.8 V; and EP633, 13.3 ± 3.2 V).