In vivo neuroprotective capacity of a Dunaliella salina extract - comprehensive transcriptomics and metabolomics study

Abstract

In this study, an exhaustive chemical characterization of a Dunaliella salina (DS) microalga extract obtained using supercritical fluids has been performed, and its neuroprotective capacity has been evaluated in vivo using an Alzheimer's disease (AD) transgenic model of Caenorhabditis elegans (strain CL4176). More than 350 compounds were annotated in the studied DS extract, with triacylglycerols, free fatty acids (FAs), carotenoids, apocarotenoids and glycerol being the most abundant. DS extract significantly protects C. elegans in a dose-dependent manner against Aβ-peptide paralysis toxicity, after 32 h, 53% of treated worms at 50 µg/mL were not paralyzed. This concentration was selected to further evaluate the transcriptomics and metabolomics changes after 26 h by using advanced analytical methodologies. The RNA-Seq data showed an alteration of 150 genes, mainly related to the stress and detoxification responses, and the retinol and lipid metabolism. The comprehensive metabolomics and lipidomics analyses allowed the identification of 793 intracellular metabolites, of which 69 were significantly altered compared to non-treated control animals. Among them, different unsaturated FAs, lysophosphatidylethanolamines, nucleosides, dipeptides and modified amino acids that have been previously reported as beneficial during AD progression, were assigned. These compounds could explain the neuroprotective capacity observed, thus, providing with new evidences of the protection mechanisms of this promising extract.

Fig. 1. Total ion current (TIC) chromatograms and the most abundant annotated compounds in Dunaliella salina (DS) extract.

a CSH-Q-TOF MS/MS ESI (-) analysis. b CSH-Q-TOF MS/MS ESI (+) analysis. c GC-Q-TOF MS analysis.

Fig. 2. Effect of Dunaliella salina (DS) extract on Caenorhabditis elegans CL4176 strain.

a Time course of the paralysis assay after Aβ-peptide induction and treated with different concentrations of DS extract (1, 10, 25 and 50 μg/mL). For each condition tested, two independent assays including n = 60 worms/assay were performed. Ginkgo biloba extract (EGb 761®) was used as a positive control extract. b Percentage of Aβ protein aggregation after 26 h of DS extract treatment (50 μg/mL) compared to control conditions (0.05% DMSO). c Percentage of intracellular reactive oxygen species (ROS) after 26 h of DS extract treatment (50 μg/mL) compared to control conditions (0.05% DMSO). In all cases, error bars indicate standard deviation of the mean.

Fig. 3. Functional enrichment analysis and pathway analysis of the differentially expressed genes observed in Caenorhabditis elegans after Dunaliella salina (DS) extract treatment (50 μg/mL, n = 5) compared to control conditions (0.05% DMSO, n = 5) for 26 h.

a Sunburst diagram obtained using WormCat 2.0 software. b Significantly enriched pathways obtained using MetaboAnalyst.

Fig. 4. Multivariate analysis and ChemRICH results of the metabolomics data from Caenorhabditis elegans experiments.

a Principal Component Analysis score plots (PC1 vs PC2) including the three groups of analyzed samples (Control, DS-Treated, Not Induced) (n = 5 for each group). b Chemical similarity enrichment results obtained from DS-Treated samples (50 μg/mL, n = 5) compared to Control conditions (0.05% DMSO, n = 5) for 26 h. The y-axis shows the most significantly altered clusters on top; the x-axis shows the XlogP values of clusters. Cluster colors give the proportion of increased or decreased compounds (red = increased, blue = decreased) in each cluster. Chemical enrichment statistics is calculated by Kolmogorov-Smirnov test. Only enrichment clusters are shown that are significantly different at p < 0.05.