Lacticaseibacillus rhamnosus HA-114 and Bacillus subtilis R0179 Prolong Lifespan and Mitigate Amyloid-β Toxicity in C. elegans via Distinct Mechanisms

Abstract

Background: Recent advances linking gut dysbiosis with neurocognitive disorders such as Alzheimer's disease (AD) suggest that the microbiota-gut-brain axis could be targeted for AD prevention, management, or treatment.

Objective: We sought to identify probiotics that can delay Aβ-induced paralysis.

Methods: Using C. elegans expressing human amyloid-β (Aβ)1-42 in body wall muscles (GMC101), we assessed the effects of several probiotic strains on paralysis.

Results: We found that Lacticaseibacillus rhamnosus HA-114 and Bacillus subtilis R0179, but not their supernatants or heat-treated forms, delayed paralysis and prolonged lifespan without affecting the levels of amyloid-β aggregates. To uncover the mechanism involved, we explored the role of two known pathways involved in neurogenerative diseases, namely mitophagy, via deletion of the mitophagy factor PINK-1, and fatty acid desaturation, via deletion of the Δ9 desaturase FAT-5. Pink-1 deletion in GMC101 worms did not modify the life-prolonging and anti-paralysis effects of HA-114 but reduced the protective effect of R0179 against paralysis without affecting its life-prolonging effect. Upon fat5 deletion in GMC101 worms, the monounsaturated C14:1 and C16:1 FAs conserved their beneficial effect while the saturated C14:0 and C16:0 FAs did not. The beneficial effects of R0179 on both lifespan and paralysis remained unaffected by fat-5 deletion, while the beneficial effect of HA-114 on paralysis and lifespan was significantly reduced.

Conclusions: Collectively with clinical and preclinical evidence in other models, our results suggest that HA-114 or R0179 could be studied as potential therapeutical adjuncts in neurodegenerative diseases such as AD.

Keywords: Age-related diseases; Alzheimer’s disease; B. subtilis R0179; Caenorhabditis elegans; L. rhamnosus HA-114; amyloid; lipid metabolism; probiotics.

Fig. 1

The effect of several probiotic strains on paralysis in GMC101 worms is strain specific. A) Representation of the scoring method for paralyzed and non-paralyzed worms. B) Paralysis assay in GMC101 worms comparing the effect of a panel of probiotic strains (10⁹ cells/mL) over 72 h following the temperature upshift (n = 3). Error bars indicate SEM. Two-way ANOVA with Dunnett’s multiple comparison test; p < 0.0001 versus OP50. C) Quantification of aggregates stained with X-34 at 24 h after the temperature upshift showing D) Aβ aggregates in the head region of GMC101 worms fed the indicated diet.

Fig. 2

Live HA-114 and R0179 confer maximal protection against paralysis. Paralysis assay in GMC101 worms comparing the effect of live HA-114 and R0179 (10⁹ cells/mL) versus their corresponding cell-free supernatants (A and C, respectively; n = 3) or probiotic strains subjected to heat treatment (HT) at 80°C for 20 min prior to seeding (B and D, respectively; n = 3). Paralysis was scored over 72 h following the temperature upshift. Error bars indicate SEM. Two-way ANOVA with Dunnett’s multiple comparison test (versus OP50); ^*p < 0.05; ^**p < 0.01.

Fig. 3

Pink-1 is not essential for the effects of HA-114 on lifespan and paralysis in GMC101 worms. Lifespan of A) N2 (n = 4), B) GMC101 (n = 4), and C) GMC101;Δpink-1 (n = 2) fed the indicated diets. Gehan-Breslow-Wilcoxon test (versus OP50); ^****p < 0.0001; ^***p < 0.001; ^**p < 0.01. D) Paralysis of GMC101;Δpink-1 fed the indicated diets (n = 9) scored over 48 h following temperature upshift. Error bars indicate SEM. Two-way ANOVA with Dunnett’s multiple comparison test (versus OP50); ^****p < 0.0001.

Fig. 4

Fatty acids typically desaturated by fat-5 are more efficient at reducing paralysis. A) Paralysis assay in OP50-fed GMC101 worms comparing the effect of the indicated saturated fatty acids (200 μM; n = 3). B) Schematics of the fatty acid desaturation pathways in C. elegans (adapted from³⁵). C) Paralysis assay in OP50-fed GMC101 worms comparing the effect of saturated and monounsaturated C14 and C16 fatty acids (200 μM; n = 6). Error bars indicate SEM. Two-way ANOVA with Dunnett’s multiple comparison test (versus OP50); ^****p < 0.0001.

Fig. 5

The effect of HA-114 on paralysis and lifespan requires fat-5. A) Paralysis assay in OP50-fed GMC101; Δfat-5 worms comparing the effect of the indicated saturated and monounsaturated C14 and C16 fatty acids (200 μM; n = 6). Error bars indicate SEM. Two-way ANOVA with Dunnett’s multiple comparison test (versus OP50); ^*p < 0.05. B) Paralysis assay in GMC101 (n = 4) and GMC101; fat-5 (n = 9) fed the indicated diets. Error bars indicate SEM. Two-way ANOVA with Tukey’s multiple comparison test; ^***p < 0.001. C) Lifespan assay in GMC101 (n = 4) and GMC101;Δfat-5 (n = 4; n = 2 for OP50) fed the indicated diets. Gehan-Breslow-Wilcoxon test (versus GMC101 corresponding diet); ^**p < 0.0001; n.s., not significant. D) Bar graph of mean lifespan (n = 3; ^*p < 0.05 versus N2/HA-114). Dashed line at 12 days.

Fig. 6

Relative abundance of selected fatty acids in OP50, R0179, and HA-114. Doughnuts showing the relative abundance of A) short-chain fatty acids and B) C14-C18 fatty acids in OP50 (inner circle), R0179 (middle circle) and HA-114 (outer circle).