Reduced Function of the Glutathione S-Transferase S1 Suppresses Behavioral Hyperexcitability in Drosophila Expressing Mutant Voltage-Gated Sodium Channels

Abstract

Voltage-gated sodium (Na_v) channels play a central role in the generation and propagation of action potentials in excitable cells such as neurons and muscles. To determine how the phenotypes of Na_v-channel mutants are affected by other genes, we performed a forward genetic screen for dominant modifiers of the seizure-prone, gain-of-function Drosophila melanogaster Na_v-channel mutant, para^Shu Our analyses using chromosome deficiencies, gene-specific RNA interference, and single-gene mutants revealed that a null allele of glutathione S-transferase S1 (GstS1) dominantly suppresses para^Shu phenotypes. Reduced GstS1 function also suppressed phenotypes of other seizure-prone Na_v-channel mutants, para^GEFS+ and para^bss Notably, para^Shu mutants expressed 50% less GstS1 than wild-type flies, further supporting the notion that para^Shu and GstS1 interact functionally. Introduction of a loss-of-function GstS1 mutation into a para^Shu background led to up- and down-regulation of various genes, with those encoding cytochrome P450 (CYP) enzymes most significantly over-represented in this group. Because GstS1 is a fly ortholog of mammalian hematopoietic prostaglandin D synthase, and in mammals CYPs are involved in the oxygenation of polyunsaturated fatty acids including prostaglandins, our results raise the intriguing possibility that bioactive lipids play a role in GstS1-mediated suppression of para^Shu phenotypes.

Keywords: Forward genetic screen; RNA-sequencing analysis; epilepsy; genetic modifiers.

Figure 1

Overlapping deficiencies in the 53E-53F chromosomal region and suppression of the climbing defect of para^Shu mutants. (A) Chromosomal deficiencies in 53E-53F (right arm of second chromosome) that were examined for effects on para^Shu phenotypes. The cytological location and chromosomal break points of each deficiency are indicated in the table. (B) Reactive climbing behaviors of para^Shu heterozygous females in the presence of the tested deficiencies. Three to eight groups of ∼20 flies per genotype were tested. The total numbers of flies tested in each group were 141 (control), 101 (Df(2R)P803-Δ15), 93 (Df(2R)Exel6065), 111 (Df(2R)BSC433), and 53 (Df(2R)Exel6066). Climbing indices are presented as box plots. The Kruskal-Wallis one-way ANOVA on ranks with Dunn’s method was used to compare between the control and deficiency groups. ***P < 0.001; NS, not significant (P > 0.05).

Figure 2

Suppression of multiple para^Shu phenotypes by deletion of the genomic region 53F4-53F8. The effects of chromosomal deficiencies Df(2R)BSC273 (49F4-50A13), Df(2R)BSC330 (51D3-51F9), and Df(2R)BSC433 (53F4-53F8) on para^Shu phenotypes were examined. (A) Frequency of morphological defects, including down-turned wings and an indented thorax. Numbers in the bar graph indicate how many flies were scored. (B) Severity of spontaneous tremors. Numbers in the boxplot indicate how many flies were scored. (C) Severity of heat-induced seizures. Three groups of 30 flies were used per genotype. Data are shown as the averages and SEM. Fisher’s exact test with Bonferroni correction (A), the Kruskal-Wallis one-way ANOVA on ranks with Dunn’s method (B), and two-way repeated measures ANOVA and Holm-Sidak multiple comparisons (C) were used for comparisons between the control and deficiency groups. ***P < 0.001; *P < 0.05; NS, not significant (P > 0.05).

Figure 3

Glutathione S-transferase S1 (GstS1) as a robust genetic modifier of para^Shu. (A) Depiction of six genes that are localized within chromosomal region 53F4-53F8 and disrupted by the chromosomal deficiency Df(2R)BSC433. Arrows indicate the direction of gene transcription. (B) The frequency of para^Shu morphological phenotypes following RNAi-mediated knockdown of each gene. Gene-specific RNAi was ubiquitously expressed using da-GAl4 in para^Shu heterozygous females (e.g., para^Shu/+; da-GAl4/UAS-RNAi). The downturned wing (Wings) and indented thorax (Thorax) phenotypes were scored. Numbers in the bar graph indicate how many flies were scored. Fisher’s exact test with Bonferroni correction was used to analyze the data. ***P < 0.001; NS, not significant (P > 0.05).

Figure 4

GstS1^M26 as a dominant suppressor of para^Shu phenotypes. The effects of the GstS1 null allele, GstS1^M26, on para^Shu phenotypes were examined in flies of three genotypes: (1) para^Shu/+; +/+, (2) para^Shu/+; GstS1^M26/+, and (3) para^Shu/+; GstS1^M26/GstS1^M26. (A) Frequencies of down-turned wings (Wings) and indented thorax (Thorax). Numbers in the bar graph indicate how many flies were scored. (B) Severity of spontaneous tremors. 8–10-day-old para^Shu/+ females were used. Numbers in the boxplot indicate how many flies were scored. (C) Frequencies of heat-induced seizures. Three groups of 30-50 flies at 4-5 days after eclosion were used per genotype. Averages are shown with SEM. Fisher’s exact test with Bonferroni correction (A), the Kruskal-Wallis one-way ANOVA on ranks with Dunn’s method, (B) and two-way repeated measures ANOVA and Holm-Sidak multiple comparisons (C) were used to analyze the data. ***P < 0.001; *P < 0.05; NS, not significant (P > 0.05).

Figure 5

Phenotypes of other neurological mutants are suppressed by GstS1^M26. (A) Frequencies of heat-induced seizure at 40°C in para^GEFS+ plus GstS1^M26 males (para^GEFS+/Y; GstS1^M26/+) or para^GEFS+ males (para^GEFS+/Y; +/+). (B) Frequencies of para^DS males that did not stand at 37°C. For (A) and (B), averages of 3 experiments and SEM are shown. In each experiment, 30 flies were examined. (C) Recovery time required for para^bss1 plus GstS1^M26 males (para^bss1/Y; GstS1^M26/+) and para^bss1 males (para^bss1/Y; +/+) to recover from paralysis induced by mechanical shock. Data are presented as box plots. Total numbers of flies observed were 127 and 223 flies for para^bss1/Y; +/+ and para^bss1/Y; GstS1^M26/+, respectively. Data analysis involved two-way repeated measures ANOVA and Holm-Sidak multiple comparisons (A and B) and the Mann-Whitney U-test (C). ***P < 0.001; *P < 0.05; NS, not significant (P > 0.05).

Figure 6

Reduction of GstS1 expression in para^Shu. Levels of GstS1 transcript, as evaluated by RNAseq analysis in control (Canton-S) and para^Shu heterozygous females with or without a GstS1^M26 mutation (para^Shu/+; +/+ or para^Shu/+; GstS1^M26/+) (see Materials and Methods). Averages of four biological replicates are shown, as normalized read counts with SEM and adjusted P-values (P_adj). ***P_adj < 0.001.