Krill oil protects dopaminergic neurons from age-related degeneration through temporal transcriptome rewiring and suppression of several hallmarks of aging

Abstract

There is accumulating evidence that interfering with the basic aging mechanisms can enhance healthy longevity. The interventional/therapeutic strategies targeting multiple aging hallmarks could be more effective than targeting one hallmark. While health-promoting qualities of marine oils have been extensively studied, the underlying molecular mechanisms are not fully understood. Lipid extracts from Antarctic krill are rich in long-chain omega-3 fatty acids choline, and astaxanthin. Here, we used C. elegans and human cells to investigate whether krill oil promotes healthy aging. In a C. elegans model of Parkinson´s disease, we show that krill oil protects dopaminergic neurons from aging-related degeneration, decreases alpha-synuclein aggregation, and improves dopamine-dependent behavior and cognition. Krill oil rewires distinct gene expression programs that contribute to attenuating several aging hallmarks, including oxidative stress, proteotoxic stress, senescence, genomic instability, and mitochondrial dysfunction. Mechanistically, krill oil increases neuronal resilience through temporal transcriptome rewiring to promote anti-oxidative stress and anti-inflammation via healthspan regulating transcription factors such as SNK-1. Moreover, krill oil promotes dopaminergic neuron survival through regulation of synaptic transmission and neuronal functions via PBO-2 and RIM-1. Collectively, krill oil rewires global gene expression programs and promotes healthy aging via abrogating multiple aging hallmarks, suggesting directions for further pre-clinical and clinical explorations.

Keywords: aging; healthspan; krill oil; mitochondrial health; senescence.

Figure 1

Krill oil promotes healthy dopaminergic neuronal aging. (A) Representative images of the head region of PD animals at day 1, day 3 and day 6 of adulthood, Scale bar, 20 μm. (B) Survival of anterior CEPs and ADEs DA neurons during aging in response to Krill oil (n = 35 nematodes per experiment; three independent experiments, s.e.m; ^***p < 0.001; one-way ANOVA followed by Bonferroni’s multiple comparison test. (C) Representative images of α-SYN aggregation in body wall muscles in day 1 and day 6 animals, Scale bar, 20 μm. (D) The number of aggregates in day 1 and day 6 animals in response to krill oil (n = 15 nematodes, s.e.m; NS and ^***p < 0.001; one-way ANOVA followed by Bonferroni’s multiple comparison test). (E) Column scatter plot representing basal slowing response of wild type and PD animals at adult day 6. Body bends per 20 second measured on NGM plates with and without bacteria (n = 30; Error bars, s.e.m; ^***p < 0.001; one-way ANOVA followed by Bonferroni’s multiple comparison test. (F) Column scatter plot representing locomotion activity wild type and PD animals at adult day 6. The activity was scored for 60 minutes (n = 50 animals per experiment; Error bars, s.e.m; NS and ^*p < 0.05; one-way ANOVA followed by Bonferroni’s multiple comparison test.

Figure 2

Krill oil delays senescence. (A, B) Representative images of β-gal staining in the head region in 9-day old adults, Scale bar, 20 μm. Column scatter plot representing the percentage of worms with positive senescence staining in three independent experiments (n = 50–100 individuals, column indicates mean, error bars, s.e.m, ^***p < 0.001; one-way ANOVA followed by Bonferroni’s multiple comparison test). (C, D) Images and quantification represents senescence in late passage BJ cells using β-gal staining (three independent experiments, Scale bar, 20 μm, Error bars, s.e.m; ^*p < 0.05; one-way ANOVA followed by Bonferroni’s multiple comparison test). (E) Relative p21 and TGFβ mRNA levels in BJ cells at passage 10 and 21 treated, or not, with Krill oil (100 μg/ml, 6 days) as measured by qPCR. Data represent means ± s.d., n = 3. ^*P ≤ 0.05, ^**P ≤ 0.01, ^****P ≤ 0.0001 (two-tailed Student’s t-test).

Figure 3

Krill oil improves mitochondrial health. (A, B) Representative image and quantification of 8-Oxo (dG) staining in old day 6 PD animals (n = 10 individuals, Error bars, s.e.m; ^****p < 0.0001; one-way ANOVA followed by Bonferroni’s multiple comparison test). (C) Oxygen consumption rate in day 1, day 3 and day 6 PD animals (n = 50 individuals, two independent experiments, Error bars, s.e.m; NS and ^*p < 0.05, ^**p < 0.01; one-way ANOVA followed by Bonferroni’s multiple comparison test). (D, E) Image and quantification of mitochondrial membrane potential measured using TMRE staining in BJ fibroblast at passage 16 (p16) in the absence and presence of krill oil (n = 6 independent experiments, Scale bar, 20 μm, Error bars, s.e.m; ^**p < 0.01; one-way ANOVA followed by Bonferroni’s multiple comparison test).

Figure 4

Krill oil alters genes regulation. (A) Differentially expressed genes (DEG) in PD animals in the presence and absence of krill oil treatment in day 1, day 3 and day 6 old animals. (B) Heat map of the time series analyses comprising all DEGs revealing 24 co-regulated clusters. (C) Examples of clusters (C10, C19, C5 and C21) with typical trajectories in PD animals. (D) Represents multi-dimensional scaling (MDS) plots showing all GOs and separating them based on clusters having similar functions.

Figure 5

Krill oil rewires gene expression to promote neuron survival. (A) Heatmap from WGCNA analysis shows the correlation values between all of the found modules (gene groups) and defined parameters (krill oil treated, DA neuron survival, OCR and locomotion). The correlation ranges from +1 (completely positively correlated) to -1 (perfect negative correlation). (B–E) Gprofiler plots with functionally enriched transcription factors indicated; (B) Representation of the modules (turquoise, white and saddlebrown) upregulated in day 1 PD animals. (C) Representation of the modules (red, pink and dark grey) downregulated in day 1 PD animals. (D) Representation of the modules (blue, tan, cyan, light cyan, violet, dark magenta, sienna) upregulated in day 3 PD animals. (E) Representation of the modules (magenta, purple, dark olive green, and yellow green) upregulated in day 6 PD animals.

Figure 6

Gene expression rewiring promotes dopaminergic neuron survival during aging. (A) Representative images of the head region of PD animals at day 6 adulthood in control and krill oil treated animals following knockdown of jnk-1, skn-1, hmg-5 and lmd-3 by RNAi, Scale bar, 20 μm. Time series plot for same genes are show on top. The scatter dot plots represent GFP intensity of the CEPs dopaminergic neurons in day 6 PD nematodes following knockdown of jnk-1, skn-1, hmg-5 and lmd-3 (n = 20 individuals; Error bars, s.e.m; ^***p < 0.001; one-way ANOVA followed by Bonferroni’s multiple comparison test). (B) representative images and scatter dot plots represents intensity of the dopaminergic neurons after depleting cnnm-3, pbo-2 and rim-1 in PD animals (n = 14 individuals, Scale bar, 20 μm Error bars, s.e.m; ^***p < 0.001; one-way ANOVA followed by Bonferroni’s multiple comparison test), co-related with time series plot for same genes.

Figure 7

Krill oil improves healthspan. (A) The learning index was calculated from a positive association with butanone in wild type and PD animals at adult day 6 (n = 150 individuals, two independent experiments, error bars, s.e.m; NS, ^*p < 0.05; one-way ANOVA followed by Bonferroni’s multiple comparison test). (B) Quantification of pharyngeal pumping frequency (Hz) monitored in wild type and PD animals using the NemaMetrix screenchip based assay. The column scatter plot represents pharyngeal pumping frequency of adult day 3 animals (n = 15–20 individuals, Error bars, s.e.m NS and ^**P ≤ 0.001, ^****p < 0.0001; one-way ANOVA followed by Bonferroni’s multiple comparison test). (C) Predicted biological age of wild type and PD animals in response to krill oil using BiTAge calculator. (D) TEM image of day 9 old in PD animals, boxed area represents the cuticle (scale bar 2000 nm).

Figure 8

Krill oil promotes neuron health by suppressing aging hallmarks. The graphical representation demonstrates the benefit of krill oil and the pathways involved which leads to protection of DA neurons in C. elegans and human BJ fibroblast cells. The illustration was generated using https://biorender.com/.