Dietary Supplementation with Milk Lipids Leads to Suppression of Developmental and Behavioral Phenotypes of Hyperexcitable Drosophila Mutants

Abstract

Dietary modifications often have a profound impact on the penetrance and expressivity of neurological phenotypes that are caused by genetic defects. Our previous studies in Drosophila melanogaster revealed that seizure-like phenotypes of gain-of-function voltage-gated sodium (Na_v) channel mutants (para^Shu, para^bss1, and para^GEFS+), as well as other seizure-prone "bang-sensitive" mutants (eas and sda), were drastically suppressed by supplementation of a standard diet with milk whey. In the current study we sought to determine which components of milk whey are responsible for the diet-dependent suppression of their hyperexcitable phenotypes. Our systematic analysis reveals that supplementing the diet with a modest amount of milk lipids (0.26% w/v) mimics the effects of milk whey. We further found that a minor milk lipid component, α-linolenic acid, contributed to the diet-dependent suppression of adult para^Shu phenotypes. Given that lipid supplementation during the larval stages effectively suppressed adult para^Shu phenotypes, dietary lipids likely modify neural development to compensate for the defects caused by the mutations. Consistent with this notion, lipid feeding fully rescued abnormal dendrite development of class IV sensory neurons in para^Shu larvae. Overall, our findings demonstrate that milk lipids are sufficient to ameliorate hyperexcitable phenotypes in Drosophila mutants, providing a foundation for future investigation of the molecular and cellular mechanisms by which dietary lipids modify genetically induced abnormalities in neural development, physiology, and behavior.

Keywords: RNA-sequencing; gene-environment interaction; neuronal development; seizure; voltage-gated sodium channel; α-linolenic acid.

Figure 1.. A biological activity in milk whey that can suppress para^Shu hyperexcitable phenotypes is associated with large molecular complexes.

(A) Schematic showing how milk whey components were separated into fractions that pass (passing, PS) and do not pass (retained, RT) through Amicon Centrifugal Filters, Ultracel-3, 30, and 100. (B and C) The effects of diets supplemented with different PS and RT fractions on para^Shu adult phenotypes: (B) down-turned wings and (C) severity of spontaneous tremors indirectly accessed by abnormal locomotor activity. For more detail on the assay system, refer to Experimental Procedures. Numbers of flies scored under each condition are indicated above each bar. Statistical analyses were performed using Fisher’s exact test with Bonferroni correction (B) and the Kruskal-Wallis one-way ANOVA on ranks with Dunn’s method (C). Statistically significant differences between the control (regular diet) and experimental groups (supplemented diets) are shown. ***P < 0.001; **P < 0.01.

Figure 2.. Multiple para^Shu adult phenotypes are suppressed by diets supplemented with milk lipid-containing fractions.

(A and B) Effects of polar and non-polar fractions separated from milk whey solution on (A) down-turned wings and (B) tremor severity in para^Shu mutants. (C and D) Effects of different fractions isolated from bovine milk on (C) down-turned wings and (D) tremor severity in para^Shu mutants. Fractions include: aqueous (AQ); enriched for milk fat globules (MFG); enriched for milk fat globule membranes (MFGM); and enriched for the triacylglycerol-rich core of the MFGs (CORE). (E) Effects of the whey and CORE fractions on heat-induced seizures in para^Shu mutants and control flies. Averages are shown with SEM. Numbers of flies scored under each condition are indicated above each bar. Statistical analyses were performed using Fisher’s exact test with Bonferroni correction (A and C), the Kruskal-Wallis one-way ANOVA on ranks with Dunn’s method (B and D), and two-way repeated measures ANOVA and Holm-Sidak multiple comparisons (E). Statistically significant differences between the controls (regular diet) and experimental groups (supplemented diets) are shown. ****P < 0.0001; ***P < 0.001; *P < 0.05.

Figure 3.. A diet supplemented with the CORE fraction reduces the severity of bang-sensitive phenotypes caused by different genetic defects.

Recovery time for three distinct bang-sensitive mutants (A) bang senseless (bss or para^bss1), (B) easily shocked (eas²), and (C) slamdance (sda) when raised on a control diet (blue) or a diet supplemented with the CORE fraction (yellow). Time required for the mutants to recover from paralysis induced by mechanical shock is shown as boxplots. Numbers of flies scored under each condition are indicated above each bar. Statistical analyses were performed using the Mann-Whitney U test. Statistically significant differences are shown. ***P < 0.001.

Figure 4.. Administration of a diet supplemented with the CORE fraction during larval stages leads to suppression of the adult para^Shu phenotypes.

Tremor severity in para^Shu mutants fed the control diet or a diet supplemented with the CORE fraction during the larval stage and/or adulthood. Feeding paradigms are indicated below the plot (control, blue; CORE-supplemented, yellow). Numbers of flies scored under each condition are indicated above each box. Statistical analyses were performed using the Kruskal-Wallis one-way ANOVA on ranks with Dunn’s method. Statistical significance of differences between the control group (fed the regular diet during both larval and adult stages) and experimental groups, and between CORE-treated groups are shown. ****P < 0.0001, ***P < 0.001.

Figure 5.. α-linolenic acid contributes to diet-dependent suppression of para^Shu phenotypes.

(A and B) Effects of modified diets on (A) down-turned wings and (B) tremor severity in para^Shu mutants. Diets include: regular diet (Cont), diet supplemented with the CORE fraction (CORE), and diets supplemented with specific fatty acids or TAG (1 mM). These include butyric acid (BA, C4:0), decanoic acid (DA, C10:0), myristic acid (MA, C14:0), palmitic acid (PA, C16:0), stearic acid (SA, C18:0), oleic acid (OA, C18:1), linoleic acid (LA, C18:2), α-linolenic acid (ALA, C18:3), and trilinolenin (TLN). (C) Effects of diets supplemented with ALA or TLN on heat-induced seizures in para^Shu mutants. (D) Effects of timing of ALA feeding on suppression of para^Shu adult tremor phenotype. Tremor severity in para^Shu mutants fed the control diet or a diet supplemented with ALA during the larval stage and/or adulthood. Feeding paradigms are indicated below the plot (control, blue; ALA-supplemented, red). Numbers of flies scored under each condition are indicated. Statistical analyses were performed using Fisher’s exact test with Bonferroni correction (A), the Kruskal-Wallis one-way ANOVA on ranks with Dunn’s method (B and D), and two-way repeated measures ANOVA and Holm-Sidak multiple comparisons (C). Statistically significant differences between controls (regular diet) and experimental groups (supplemented diets) are shown. ****P < 0.0001, ***P < 0.001; **P < 0.01; *P < 0.05.

Figure 6.. Increased proximal dendritic branching of C4da neurons associated with para^Shu hyperexcitability is suppressed by dietary lipid supplementation.

(A, C, and F) Representative dendritic traces of larval class IV dendritic arborization (C4da) neurons expressing ppkCD4tdGFP in the presence or absence of dietary lipid supplementation. Proximal somatodendritic regions within a 100 μm radius and soma position are indicated by a red circle and red star respectively. (A) +/+; ppkCD4tdGFP/+ (No lipid); (C) para^Shu/+; ppkCD4tdGFP/+ (No lipid); (F) para^Shu/+; ppkCD4tdGFP/+ (Plus lipid). (B) Sholl analysis of C4da dendritic branching +/− dietary lipid supplementation indicates increased dendritic complexity in regions proximal to the soma. (D) Dendritic complexity represented by area under the Sholl curve at radii >150 μm is not significantly different between control (+/+) and para^Shu neurons +/− dietary lipids (AUC, panel D). (E) Dendritic complexity (AUC) is increased in the proximal somatodendritic regions of para^Shu neurons between 10–150 μm radii relative to wild-type controls. This increase in AUC (10–150 μm) is suppressed in para^Shu neurons from larvae receiving dietary lipid supplementation. (G and H) Increased dendritic branch formation in proximal regions (<100 μm radius) of para^Shu mutant neurons is also reflected as significantly increased branch numbers (G) and junction number (H). Both values representative of increased branch formation are suppressed by dietary lipid supplementation. n=12 for all genotypes; ****P < 0.0001, one-way ANOVA with Tukey post-test.

Figure 7.. In para^Shu mutants, the expression levels of four genes are altered in the same directions by a GstS1 mutation and supplementation of the diet with milk lipids.

(A) Numbers of genes that are differentially expressed in para^Shu mutants in response to the presence of the GstS1^M26 mutation and to a diet supplemented with the CORE fraction. (B) Transcript levels of Act88F, pHCl-2, Gld, and Cpr47Ef, as assessed by RNA-seq analysis in four biological replicates of the following treatment groups: Canton-S flies fed a regular diet, para^Shu mutants fed a regular diet, para^Shu mutants fed a diet supplemented with lipids, and para^Shu; GstS1^M26 double mutants fed a regular diet. Averages of normalized read counts are shown, with SEM. Statistical analyses were performed using one-way ANOVA with Tukey post-test. Statistically significant differences are shown. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05.