Docosahexaenoic Acid (DHA) Supplementation in a Triglyceride Form Prevents from Polyglutamine-Induced Dysfunctions in Caenorhabditis elegans

Abstract

A common hallmark of neurodegenerative diseases is the accumulation of polypeptide aggregates in neurons. Despite the primary cause of these diseases being inherently genetic, their development can be delayed with proper preventive treatments. Long-chain polyunsaturated fatty acids (ω-3 LCPUFA) are promising bioactive nutrients that are beneficial for brain health. In this study, the impact of an oil rich in a structured form of docosahexaenoic acid (DHA) triglyceride (TG) was assessed in a Caenorhabditis elegans model expressing long poly-glutamine (polyQ) chains, which mimics the symptomatology of polyQ-related neurodegenerative diseases such as Huntington's disease (HD), among others. The lifespan, the motility, the number of polyQ aggregates, the oxidative stress resistance, and the cognitive performance associated with sensitive stimuli was measured in mutant nematodes with polyQ aggregates. Overall, DHA-TG at 0.5 µM improved the lifespan, the motility, the oxidative stress resistance, and the cognitive performance of the nematodes, emphasizing the protection against serotonergic synapse dysfunction. Furthermore, the treatment reduced the polyQ aggregates in the nematodes. The data described herein shed light on the connection between DHA and the cognitive performance in neurodegenerative diseases and demonstrated the potential of DHA-TG as nutritional co-adjuvant to prevent the development of polyQ-associated dysfunctions.

Keywords: C. elegans; DHA; Huntington’s; omega-3; polyQ; serotonin.

Figure 1

Healthspan effect of DHA-TG in N2 wild-type (WT) nematodes. (A) Effect on the lifespan at 20 °C. (B) Effect in motility measuring thrashes per minute at the fertile stage (4-day adult) and the aged stage (12-day adult) after treatments; data are represented as boxes showing the median of thrashing, Q1 and Q3 quartiles, and whiskers expressing minimum to maximum values (n > 30 for each condition). The statistical differences compared to the vehicle were considered significant at p < 0.05 (*) after a two-way ANOVA followed by Dunnett’s post-test.

Figure 2

Healthspan effect of DHA-TG in nematode strain AM101 (rmIs110[F25B3.3p::Q40::YFP]). (A) Effect on the lifespan at 20 °C. Statistical differences in the lifespan were considered significant at p < 0.01 (**) and p < 0.001 (***) after a Long-rank test of the survival curves vs. the 40Q vehicle. p-value is expressed beside the curves (n.s. = not significant). (B) Effect in motility measuring thrashes per minute of nematodes at 4-day adult stage after treatments; the data are represented as boxes showing the median of thrashing, Q1 and Q3 quartiles, and whiskers expressing the minimum to maximum values (n > 30 for each condition). Statistical differences compared to the 40Q vehicle were considered significant at p < 0.01 (**) after one-way ANOVA followed by Tukey’s test (n.s. = not significant).

Figure 3

(A) Pictures of AM141 nematodes (rmIs133 [unc-54p::Q40::YFP]) on the 4th day of adulthood after treatments. Worms were mounted onto 2% agar pads and anesthetized with a drop of 0.5 M sodium azide. Images were taken using a DM2500 (Leica, Wetzlar, Germany) vertical fluorescence microscope. (B) Number of 40Q aggregates in the AM141 rmIs133[unc-54p::Q40::YFP]) strain nematodes at 4 days of adulthood after treatments. Data shows a line expressing the average of 40Q aggregates per picture ±SD counted in 4 different pictures. Each point (4 points per treatment) represents an average of 40Q aggregates per picture. Each picture had 5 worms for a total n = 20. Fluorescent 40Q aggregates were counted with ImageJ. Differences were considered significant at p < 0.01 (**) and p < 0.001 (***) after one-way ANOVA followed by Tukey’s test.

Figure 4

Expression levels of aak-2 in AM101 mutant nematode at 4 days of adulthood after DHA-TG 0.5µM treatment expressed vs. WT. statistical differences compared to WT vehicle are expressed with (*), and statistical differences compared to 40Q vehicle are expressed as (#). Differences were considered significant at p < 0.001 (***) and p < 0.05 (#) after one-way ANOVA followed by Tukey’s test.

Figure 5

Stress resistance capacity of nematode strain AM101 (rmIs110[F25B3.3p::Q40::YFP]) after DHA-TG 0.5µM treatment. (A) Survival curves at 20 °C inducing ROS with 50 mM PQ solution (n > 30 for each condition). Statistical differences considered significant at p < 0.05 (*) and p < 0.001 (***) 8 after Long-rank test of survival curves. p-value and tests are detailed beside curves (n.s. = not significant). (B) Expression levels of sod-3 in AM101 mutant nematodes at 4 days of adulthood expressed vs. WT. statistical differences compared to WT vehicle are expressed with (*), and statistical differences compared to 40Q vehicle are expressed as (#). Differences were considered significant at p < 0.05 (#) and p < 0.001 (***) after one-way ANOVA followed by Tukey’s test.

Figure 6

Dopaminergic response of nematode strain AM101 (rmIs110[F25B3.3p::Q40::YFP]) at 4 days of adulthood after DHA-TG 0.5 µM treatment. (A) BSR represented as Δbody bends per 20 s. The data are represented as boxes showing the median of Δbody bends, Q1 and Q3 quartiles, and whiskers expressing the minimum to maximum values (n > 12 for each condition). Statistical differences compared to WT vehicle were considered significant at p < 0.05 (*) and p < 0.01 (**) after one-way ANOVA followed by Tukey’s test. (B) Expression levels of dat-1 in AM101 mutant nematodes expressed vs. WT. statistical differences compared to WT vehicle are expressed with (*), and statistical differences compared to 40Q vehicle are expressed as (#). Differences were considered significant at p < 0.05 (#) and p < 0.01 (**) after one-way ANOVA followed by Tukey’s test.

Figure 7

Serotonergic response of nematode strain AM101 (rmIs110[F25B3.3p::Q40::YFP]) at 4 days of adulthood after DHA-TG 0.5 µM treatment. (A) ESR represented as Δbody bends per 20 s. The data are represented as boxes showing the median of Δbody bends, Q1 and Q3 quartiles, and whiskers expressing the minimum to maximum values (n > 12 for each condition). Statistical differences compared to WT vehicle were considered significant at p < 0.05 (*) after one-way ANOVA followed by Tukey’s test (n.s. = not significant). (B) Gene expression levels of ser4 in AM101 strain vs. WT. (C) Gene expression levels of mod5 in AM101 strain vs. WT. (D) Gene expression levels of tph1 in AM101 strain vs. WT. In (B–D) statistical differences compared to WT vehicle were considered significant at p < 0.05 (*) and p < 0.001 (***) after one-way ANOVA followed by Tukey’s test (n.s. = not significant).

Figure 8

Effects of DHA-TG on nematode’s mutant strains with polyQ aggregates.