Downregulation of oxidative stress-mediated glial innate immune response suppresses seizures in a fly epilepsy model

Abstract

Previous work in our laboratory has shown that mutations in prickle (pk) cause myoclonic-like seizures and ataxia in Drosophila, similar to what is observed in humans carrying mutations in orthologous PRICKLE genes. Here, we show that pk mutant brains show elevated, sustained neuronal cell death that correlates with increasing seizure penetrance, as well as an upregulation of mitochondrial oxidative stress and innate immune response (IIR) genes. Moreover, flies exhibiting more robust seizures show increased levels of IIR-associated target gene expression suggesting they may be linked. Genetic knockdown in glia of either arm of the IIR (Immune Deficiency [Imd] or Toll) leads to a reduction in neuronal death, which in turn suppresses seizure activity, with oxidative stress acting upstream of IIR. These data provide direct genetic evidence that oxidative stress in combination with glial-mediated IIR leads to progression of an epilepsy disorder.

Keywords: CP: Immunology; CP: Neuroscience; epilepsy; glia; innate immunity; neurodegeneration; neuronal cell death; oxidative stress; prickle; seizure disorder.

Figure 1.. Expressing pk^sple in neurons, but not glia, is sufficient to suppress seizures in homozygous pk^sple mutants

(A) Spontaneous seizure quantification in 15 dpe males and females of indicated genotypes reveals significant reduction in seizures when pk^sple is expressed ubiquitously. n = 5–8/genotype. (B) Spontaneous seizure quantification in 15 dpe males and females of indicated genotypes reveals significant reduction in seizures when pk^sple is expressed pan-neuronally. n = 3–5/genotype. (C) Spontaneous seizure quantification in 15 dpe males and females of indicated genotypes reveals no significant reduction in seizures when pk^sple is expressed in glial cells. n = 4–5/genotype. Data are shown as mean ± SEM; one-way ANOVA with Holm-Sidak multiple comparisons test. n = number of seizure assays performed, 8–10 flies/assay. **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. ns, no significant difference. For source data, see Data S3.

Figure 2.. pk^sple mutant brains show significant upregulation of innate immune response pathway genes across adulthood

(A) Volcano plot of microarray analysis of 7–10 dpe sple/sple brains compared with controls. log₁₀ p values (y axis) were plotted against log₂ fold changes (x axis); p < 0.05 and fold changes of ≤0.66 and ≥ 1.5 were used to call genes as being significantly downregulated or upregulated, respectively. Top Gene Ontology categories: innate immune response (green), oxidation-reduction process (blue), flavonoid biosynthesis process (orange), and heat shock response (magenta). (B) Gene Ontology terms based on enrichment scores (as in A) are listed in descending order: innate immune response, oxidation-reduction process, flavonoid biosynthetic process, and heat shock response. (C) qRT-PCR of 7–10 dpe sple/sple brains showing log₂ fold-change values of specific AMP genes relative to control. All AMP genes show significant increase in expression in sple/sple brains. Data are shown as mean ± SEM; Student’s t test. n = 3 biological replicates/genotype. (D) qRT-PCR of 7–10 dpe control, sple/sple brains from flies with no seizure penetrance, and sple/sple brains from flies with high seizure penetrance. Most AMPs show significantly higher expression in sple/sple mutants with high seizures compared with control or sple/sple mutants that had no seizures, whereas the level of expression of AMP genes was either unchanged or significantly lower in sple/sple mutants with no seizures relative to control. Data are shown as mean ± SEM; one-way ANOVA with Tukey’s multiple comparisons test. n = 3 biological replicates/group. (E) Representative confocal images and quantification of Attacin-GFP puncta (shown in green) normalized to controls in both +/+ and sple/sple mutant brains aged to 0, 5, 15, and 30 dpe show significant increase in Attacin-GFP puncta in sple/sple brains for all time points. Data shown are mean ± SEM; Student’s t test. Dots represent individual values. n = 6–17 brains/genotype. Scale bars, 50 μm. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. For source data, see Data S3.

Figure 3.. Glial cells are the source of AMP expression in pk^sple mutant brains

(A) Representative confocal images of 15 dpe +/+;Attacin-GFP and sple/sple;Attacin-GFP optic lobes expressing mCherry in glia using repo-Gal4 driver and immunostained for GFP (green), mCherry (magenta), and neuronal marker Elav (blue). Colocalization of GFP with mCherry is indicated by white asterisks, whereas colocalization of GFP with Elav is indicated by orange arrowheads. Scale bars, 5 μm. (B) Quantification of percentage of Attacin-GFP puncta colocalizing with either Elav or mCherry reveals that most puncta overlap with mCherry (glia) in sple/sple brains. Data are shown as mean ± SEM; Mann-Whitney test. n = 19 confocal sections/group. (C) Representative confocal images of optic lobe regions of +/+, +/+;repo-Gal4/UAS-RelRNAi, sple/sple, and sple/sple;repo-Gal4/UAS-RelRNAi expressing Attacin-GFP and immunostained for GFP in green. A decrease in GFP staining is observed in the brains of sple/sple mutants when Relish is knocked down specifically in glia. Scale bars, 20 μm. (D) Quantification of Attacin-GFP puncta normalized to control in +/+, +/+;repo-Gal4/UAS-RelRNAi, sple/sple, and sple/sple;repo-Gal4/UAS-RelRNAi shows significant decrease in Attacin-GFP puncta in sple/sple brains when Relish is knocked down specifically in glia. Data are shown as mean ± SEM; Kruskal-Wallis with Dunn’s multiple comparison test. Dots represent individual values. n = 6–8 brains/genotype. (E) Schematic diagram of a Drosophila brain highlighting the region (magenta box) used for obtaining representative confocal images shown in (A) and (C). *p ≤ 0.05, ***p ≤ 0.001. For source data, see Data S3.

Figure 4.. pk^sple mutants have sustained neuronal cell death and exacerbation of seizures with age

(A and B) Representative confocal images of 5 dpe +/+ (A) and sple/sple (B) adult brains stained for apoptosis marker Dcp-1 (green). Scale bars, 100 μm. (C) Quantification of Dcp-1 puncta in (A) and (B) normalized to 5 dpe controls reveals significant increase in neuronal cell death in sple/sple brains. Data are shown as mean ± SEM; Mann-Whitney test. Dots represent individual values. n = 11–12 brains/genotype. (D and E) Representative confocal images of 15 dpe +/+ (D) and sple/sple (E) brains stained for Dcp-1 (green). Scale bars, 100 μm. (F) Quantification of Dcp-1 puncta in (D) and (E) normalized to 15 dpe controls reveals significant increase in neuronal cell death in sple/sple brains. Data are shown as mean ± SEM; Mann-Whitney test. Dots represent individual values. n = 13–15 brains/genotype (G and H) Representative confocal images of 30 dpe +/+ (G) and sple/sple (H) brains stained for Dcp-1 (green). Scale bars, 100 μm. (I) Quantification of Dcp-1 puncta in (G) and (H) normalized to 30 dpe controls reveals significant increase in neuronal cell death in sple/sple brains. Data are shown as mean ± SEM; Mann-Whitney test. Dots represent individual values. n = 14 brains/genotype. (J) Representative confocal images of 15 dpe sple/sple brains immunostained for Dcp-1 (green), glial marker Repo (magenta), and neuronal marker Elav (blue). Colocalization of Dcp-1 puncta with Repo and Elav is indicated by white asterisks and orange arrowheads, respectively. Scale bars, 20 μm. (K) Quantification of percentage of Dcp-1 puncta in sple/sple brains colocalizing with either Repo or Elav. A significantly higher percentage of Dcp-1 puncta colocalize with Elav. Data are shown as mean ± SEM; Student’s t test. Dots represent individual values. n = 8 brains/group. (L) Schematic diagram of Drosophila brain highlighting the region (red box) used for obtaining representative confocal images in (J). (M) Representative confocal images of third instar larval brains of +/+ and sple/sple mutants stained for Dcp-1 in green, Elav in magenta, and merged image of Dcp-1 and Elav. Scale bars, 100 μm. (N) Quantification of Dcp-1 puncta in third instar larval brains normalized to controls shows significant increase in neuronal cell death in sple/sple larval brains. Data are shown as mean ± SEM; Student’s t test. Dots represent individual values. n = 8–10 brains/genotype. (O) Quantification of spontaneous seizures in males and females of sple/sple mutants aged to 0, 5, 15, and 30 dpe. Both male and female sple/sple mutants show an increase in spontaneous seizures across adulthood. Data are shown as mean ± SEM; one-way ANOVA with Tukey’s multiple comparisons test. n = 3–9 seizure assays/group, 8–10 flies/assay. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. For source data, see Data S3.

Figure 5.. Inhibition of glial IIR in pk^sple mutants leads to significant suppression of neuronal cell death, which in turn suppresses seizure exacerbation

(A) Quantification of Dcp-1 puncta in brains normalized to controls (+/+) reveals significant suppression in neuronal cell death in sple/sple;elav-Gal4/UAS-DIAP1 brains when compared with sple/sple, sple/sple;elav-Gal4/+, and sple/sple;UAS-DIAP1/+. sple/sple mutant brains also show significant increase in Dcp-1 puncta when compared with +/+. Data are shown as mean ± SEM; Kruskal-Wallis with Dunn’s multiple comparisons test. Dots represent individual values. n = 23–31 brains/genotype. (B) Quantification of spontaneous seizures in 15 dpe males and females of indicated genotypes reveals significant reduction in spontaneous seizures when DIAP1 is overexpressed specifically in neurons of sple/sple mutants. Data are shown as mean ± SEM; one-way ANOVA with Dunnett’s multiple comparisons test. n = 5–12 seizure assays/group, 8–10 flies/assay. (C) qRT-PCR of 7–10 dpe +/+, sple/sple, and sple/sple;elav-Gal4/UAS-DIAP1 mutant brains showing log₂ fold-change values of specific AMP genes relative to control. Most AMP genes show significant downregulation in sple/sple;elav-Gal4/UAS-DIAP1 brains relative to sple/sple brains. Data are shown as mean ± SEM; one-way ANOVA test with Tukey’s multiple comparisons test. n = 4 biological replicates/genotype. (D) Quantification of Dcp-1 puncta in brains normalized to controls (+/+) reveals significant suppression in neuronal cell death in sple/sple;repo-Gal4/UAS-RelRNAi brains when compared with sple/sple, sple/sple;repo-Gal4/+, and sple/sple;UAS-RelRNAi/+. sple/sple mutant brains also show significant increase in Dcp-1 puncta when compared with +/+. Data are shown as mean ± SEM; Kruskal-Wallis with Dunn’s multiple comparisons test. Dots represent individual values. n = 21–28 brains/genotype. (E) Quantification of spontaneous seizures in 15 dpe males and females of +/+, sple/sple, sple/sple;repo-Gal4/+, sple/sple;UAS-RelRNAi/+, and sple/sple;repo-Gal4/UAS-RelRNAi shows significant reduction in spontaneous seizures when Relish is knocked down specifically in glia. Data are shown as mean ± SEM; one-way ANOVA with Dunnett’s multiple comparisons test. n = 8–11 seizure assays/group, 8–10 flies/assay. (F) qRT-PCR of 7–10 dpe +/+, sple/sple, and sple/sple;repo-Gal4/UAS-RelRNAi mutant brains showing log₂ fold-change values of specific AMP genes relative to control. Most AMP genes show significant downregulation in sple/sple;repo-Gal4/UAS-RelRNAi brains relative to sple/sple mutant brains. Data are shown as mean ± SEM; one-way ANOVA test with Tukey’s multiple comparisons test. n = 4 biological replicates/genotype. (G) Quantification of Dcp-1 puncta in brains normalized to controls (+/+) reveals significant suppression in neuronal cell death in sple/sple;repo-Gal4/UAS-DifRNAi brains when compared with sple/sple, sple/sple;repo-Gal4/+, and sple/sple;UAS-DifRNAi/+. sple/sple brains also show significant increase in Dcp-1 puncta when compared with +/+. Data are shown as mean ± SEM; one-way ANOVA with Dunnett’s multiple comparisons test. Dots represent individual values. n = 15–19 brains/genotype. (H) Quantification of spontaneous seizures in 15 dpe males and females of +/+, sple/sple, sple/sple;repo-Gal4/+, sple/sple;UAS-DifRNAi/+, and sple/sple;repo-Gal4/UAS-DifRNAi reveals significant reduction in spontaneous seizures when Dif is knocked down specifically in glia. Data are shown as mean ± SEM; one-way ANOVA with Dunnett’s multiple comparisons test. n = 5–7 seizure assays/group, 8–10 flies/assay. (I) qRT-PCR of 7–10 dpe +/+, sple/sple, and sple/sple;repo-Gal4/UAS-DifRNAi mutant brains showing log₂ fold-change values of specific AMP genes relative to control. Four AMP genes show significant downregulation in sple/sple;repo-Gal4/UAS-DifRNAi brains relative to sple/sple brains. Data are shown as mean ± SEM; one-way ANOVA test with Tukey’s multiple comparisons test. n = 4 biological replicates/genotype. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. See Figure S5 for representative images. For source data, see Data S3.

Figure 6.. pk^sple mutant brains show increased neuronal oxidative stress that contributes to an increase in innate immune response, neuronal cell death, and seizure progression

(A and B) Representative confocal live images of +/+ (A) and sple/sple (B) larval brains expressing mitochondrial ROS GFP reporter (mt-roGFP2-Orp1). (C) Quantification of mitochondrial ROS-activated fluorescent intensity normalized to control reveals significant increase in fluorescent intensity in sple/sple larval brains. (D and E) Representative confocal live images of 0 dpe +/+ (D) and sple/sple (E) adult brains expressing mitochondrial ROS GFP reporter. (F) Quantification of mitochondrial ROS-activated fluorescent intensity normalized to control reveals no significant increase in overall fluorescent intensity in 0 dpe sple/sple brains. (G and H) Representative confocal live images of 5 dpe +/+ (G) and sple/sple (H) adult brains expressing mitochondrial ROS GFP reporter. (I) Quantification of mitochondrial ROS-activated fluorescent intensity normalized to control reveals significant decrease in fluorescent intensity in 5 dpe sple/sple brains. For (C), (F), and (I), data are shown as mean ± SEM; Student’s t test. n = 4–8 brains/genotype. **p ≤ 0.01. Scale bars, 100 μm. (J) Quantification of Dcp-1 puncta in brains normalized to controls (+/+) shows significant suppression in neuronal cell death in sple/sple;elav-Gal4/UAS-Sod1 brains when compared with sple/sple, sple/sple;elav-Gal4/+, and sple/sple;UAS-Sod1/+. sple/sple brains also show a significant increase in Dcp-1 puncta when compared with +/+. Data are shown as mean ± SEM; Kruskal-Wallis with Dunn’s multiple comparisons test. Dots represent individual values. n = 24–28 brains/genotype. See Figure S6 for representative images. (K) Quantification of spontaneous seizures in 15 dpe males and females of indicated genotypes shows significant reduction in spontaneous seizures when Sod1 is overexpressed specifically in neurons of sple/sple mutants. Data are shown as mean ± SEM; one-way ANOVA with Dunnett’s multiple comparisons test. n = 5–9 seizure assays/group, 8–10 flies/assay. (L) qRT-PCR of 7–10 dpe +/+, sple/sple, and sple/sple;elav-Gal4/UAS-Sod1 mutant brains showing log₂ fold-change values of specific AMP genes relative to control. Most AMP genes show significant downregulation in sple/sple;elav-Gal4/UAS-Sod1 brains relative to sple/sple brains. Data are shown as mean ± SEM; one-way ANOVA test with Tukey’s multiple comparisons test. n = 4 biological replicates/genotype. For source data, see Data S3.