Commensal bacteria exacerbate seizure-like phenotypes in Drosophila voltage-gated sodium channel mutants

Abstract

Mutations in voltage-gated sodium (Na_v) channels, which are essential for generating and propagating action potentials, can lead to serious neurological disorders, such as epilepsy. However, disease-causing Na_v channel mutations do not always result in severe symptoms, suggesting that the disease conditions are significantly affected by other genetic factors and various environmental exposures, collectively known as the "exposome". Notably, recent research emphasizes the pivotal role of commensal bacteria in neural development and function. Although these bacteria typically benefit the nervous system under normal conditions, their impact during pathological states remains largely unknown. Here, we investigated the influence of commensal microbes on seizure-like phenotypes exhibited by para^Shu-a gain-of-function mutant of the Drosophila Na_v channel gene, paralytic. Remarkably, the elimination of endogenous bacteria considerably ameliorated neurological impairments in para^Shu. Consistently, reintroducing bacteria, specifically from the Lactobacillus or Acetobacter genera, heightened the phenotypic severity in the bacteria-deprived mutants. These findings posit that particular native bacteria contribute to the severity of seizure-like phenotypes in para^Shu. We further uncovered that treating para^Shu with antibiotics boosted Nrf2 signaling in the gut, and that global Nrf2 activation mirrored the effects of removing bacteria from para^Shu. This raises the possibility that the removal of commensal bacteria suppresses the seizure-like manifestations through augmented antioxidant responses. Since bacterial removal during development was critical for suppression of adult para^Shu phenotypes, our research sets the stage for subsequent studies, aiming to elucidate the interplay between commensal bacteria and the developing nervous system in conditions predisposed to the hyperexcitable nervous system.

Keywords: Drosophila melanogaster; Nrf2; commensal bacteria; gut‐brain connection; seizure; voltage‐gated sodium channel.

FIGURE 1

Administration of antibiotics to para ^Shu mutants suppresses morphological and behavioral defects. (A) Representative images of vehicle‐treated (left) and TAK‐treated (right) para ^Shu gut homogenates cultured on MRS agar. Incidence of (B) downturned wings and (C) indented thorax in para ^Shu mutants treated with vehicle or antibiotics (TAK). (D) Percentage of time spent in the center area of the chamber for para ^Shu mutants treated with vehicle or TAK. (E) The average percentages of flies that seized at each time point (±SEM) at 37 °C following treatment with vehicle or TAK. The number of flies scored under each condition is indicated in italics. ***p < 0.001; NS, not significant (p > 0.05).

FIGURE 2

Germ‐free para ^Shu mutants exhibit improvements in wing morphology and seizure behavior. (A) Representative images of mock‐treated (Mock, left) and dechorionated germ‐free (Dch, right) para ^Shu gut homogenates cultured on MRS agar shows complete removal of the gut microbiota. (B) Incidence of downturned wings and (C) indented thorax in germ‐free para ^Shu mutants compared with mock‐treated mutants. (D) Percentage of time spent in the center area of the chamber for mock‐treated and germ‐free para ^Shu females. (E) The average percentages of flies seizing at each time point (±SEM) at 37 °C. The number of flies scored under each condition is indicated in italics. ***p < 0.001; NS, not significant (p > 0.05).

FIGURE 3

The removal of bacteria has different effects on neurological phenotypes in other Na_v channel mutants. (A) The average percentages of para ^GEFS+ or (B) para ^DS Na_v channel mutants not standing at each time point (±SEM) at the indicated temperatures following vehicle or TAK treatment. The number of flies scored under each condition is indicated in italics. ***p < 0.001; NS, not significant (p > 0.05).

FIGURE 4

Mono‐association of Lactobacillus or Acetobacter with para ^Shu mutants increases the severity of seizure‐like phenotypes. (A) Representative images of gut homogenates from gnotobiotic flies mono‐associated with specific gut microbes. (B) Incidence of downturned wings in germ‐free mutants and gnotobiotic para ^Shu flies mono‐associated with Lactobacillus (Lac), Acetobacter (Acet), or Candida (Can). (C) Percentage of time spent in the center area of the chamber for gnotobiotic and germ‐free flies. (D) The average percentages of flies seizing at each time point (±SEM) at 37 °C. The number of flies scored under each condition is indicated in italics. *p < 0.05; **p < 0.01; ***p < 0.001; NS, not significant (p > 0.05).

FIGURE 5

GstD1‐GFP fluorescence is increased in the gut of para ^Shu mutants fed antibiotics. (A) Representative images of GstD1‐GFP fluorescence in adult midguts dissected from CS (Ctrl) and para ^Shu flies cultured in vehicle or antibiotic‐containing food. (B) Relative fluorescence intensity in midguts dissected from CS (Ctrl) and para ^Shu females carrying the GstD1‐GFP transgene following treatment with vehicle or TAK. n = 7 replicates, 10 guts/replicate. *p < 0.05; ***p < 0.001; NS, not significant (p > 0.05).

FIGURE 6

Morphological and behavioral phenotypes in para ^Shu mutants are suppressed by a Keap1 null allele. (A) Schematic illustration of Drosophila Keap1 locus with exons depicted as rectangles and coding segments in dark color (adapted from ⁴² ). A null allele (Keap1 ⁰³⁶ ) removes 1500 bp of the Keap1 locus. (B) Incidence of downturned wings in para ^Shu mutants with a copy of the Keap1 ⁰³⁶ allele compared with mutant controls (para ^Shu /w). (C) Percentage of time spent in the center area of the chamber for para ^Shu /w and para ^Shu /w;; Keap1 ⁰³⁶ /+. (D) Average percentages of flies seizing at each time point (±SEM) at 37 °C. The number of flies scored under each condition is indicated in italics. **p < 0.01; ***p < 0.001.

FIGURE 7

Suppression of para ^Shu phenotypes requires antibiotic treatment during the larval stage. (A) Incidence of down‐turned wings in para ^Shu flies administered antibiotics during the larval stage or adult stage compared with vehicle‐fed controls. (B) Percentage of time spent in the center area of the chamber for mutants fed vehicle, TAK during the larval stage, or TAK during the adult stage. (C) The average percentages of flies seizing at each time point (±SEM) at 37°C. The total number of flies scored under each condition is indicated in italics. **p < 0.01; ***p < 0.001; NS, not significant (p > 0.05).