Lipidomics and H2(18)O labeling techniques reveal increased remodeling of DHA-containing membrane phospholipids associated with abnormal locomotor responses in α-tocopherol deficient zebrafish (danio rerio) embryos

Abstract

We hypothesized that vitamin E (α-tocopherol) is required by the developing embryonic brain to prevent depletion of highly polyunsaturated fatty acids, especially docosahexaenoic acid (DHA, 22:6), the loss of which we predicted would underlie abnormal morphological and behavioral outcomes. Therefore, we fed adult 5D zebrafish (Danio rerio) defined diets without (E-) or with added α-tocopherol (E+, 500mg RRR-α-tocopheryl acetate/kg diet) for a minimum of 80 days, and then spawned them to obtain E- and E+ embryos. The E- compared with E+ embryos were 82% less responsive (p<0.01) to a light/dark stimulus at 96h post-fertilization (hpf), demonstrating impaired locomotor behavior, even in the absence of gross morphological defects. Evaluation of phospholipid (PL) and lysophospholipid (lyso-PL) composition using untargeted lipidomics in E- compared with E+ embryos at 24, 48, 72, and 120hpf showed that four PLs and three lyso-PLs containing docosahexaenoic acid (DHA), including lysophosphatidylcholine (LPC 22:6, required for transport of DHA into the brain, p<0.001), were at lower concentrations in E- at all time-points. Additionally, H2(18)O labeling experiments revealed enhanced turnover of LPC 22:6 (p<0.001) and three other DHA-containing PLs in the E- compared with the E+ embryos, suggesting that increased membrane remodeling is a result of PL depletion. Together, these data indicate that α-tocopherol deficiency in the zebrafish embryo causes the specific depletion and increased turnover of DHA-containing PL and lyso-PLs, which may compromise DHA delivery to the brain and thereby contribute to the functional impairments observed in E- embryos.

Keywords: Brain; Development; Docosahexaenoic acid; H(2)(18)O; Mass spectrometry; Peroxidation; Phospholipids; Vitamin E.

Graphical abstract

Fig. 1

E− embryos have morphological defects and increased mortality compared to E+ embryos. Representative pictures from the two diet groups at 120 hours post-fertilization (hpf). A. Uninflated swim bladders in elsewise morphologically normal E− embryos that survived to this stage. B. An E+ embryo provided for comparison. Morphological evaluations were made using the zebrafish acquisition and analysis program (ZAAP). Phenotypic differences in E− embryos became evident between 48 and 72 hpf, with most mortalities occurring by 72 hpf. By 120 hpf, 75–88% of E− embryos from each spawn were malformed or dead.

Fig. 2

E− embryos have impaired behavior compared to E+ embryos when assessed using a locomotor response assay. Embryos were analyzed in 96-well plates, one embryo per well with 128 embryos per diet condition. Locomotor activity following a series of light/dark stimuli (one stimulus every 3 min for 24 min total) was measured as movement (mm) over time (seconds); criteria for statistical significance between conditions was an area-under-curve (AUC) difference of >30% and p<0.05 (Kolmogorov-Smirnov test). At 96 hpf, E- embryos (red) were 82% less responsive to the light than were E+ embryos (blue) (E- AUC:459; E+ AUC:2468; p<0.01). Dead or malformed embryos were removed prior to data analysis (apparently normal embryos: n=54 E−; n=94 E+). The abnormal behavioral response in the E− embryos is indicative of underlying perturbations in neurological processes, as there were no observed differences in swimming behavior between E− and E+ conditions to suggest motor impairments in the former. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

E− and E+ embryos display significantly different PL and lyso-PL composition profiles during development. Heatmaps of identified PL or lyso-PL are shown. Sample intensities were normalized against an internal standard (DT-PC 13:0/13:0) and then scaled with Pareto scaling. Figures were generated using Metabolanalyst software. Heatmap trends (high [red], low [blue]) indicate that the E− embryos contain, overall, greater quantities of lyso-PL species early in development (24 hpf) when compared to E+ embryos, but many of these species, notably those containing DHA (22:6), are markedly depleted by 120 hpf. PL trends are highly varied; however, E− embryos contain lesser quantities of PL species with DHA (22:6). A. PC, phosphatidylcholine; B. LPC, lysophosphatidylcholine; C. PE, phosphatidylethanolamine; D. PI, phosphatidylinositol and PS, phosphatidylserine E. LPE, lysophosphatidylethanolamine; LPI, lysophosphatidylinositol and LPS, lysophosphatidylserine.

Fig. 4

Four specific PLs containing DHA (22:6) are significantly lower in E− embryos. Lipidomic analysis of lipid extracts from E− and E+ embryo samples (n=15/sample; 4 samples/group) taken at 24, 48, 72, and 120 hpf. Lipid species were confirmed by high-resolution MS, MS/MS fragmentation, and isotopic distribution, and then compared using the PeakView database. Peak intensities were used for relative quantification between E− and E+ conditions. The data shown are means±SEM. Two-way ANOVA, Bonferroni's post-test for multiple comparisons (**p<0.01, ***p<0.001, ****p<0.0001).

Fig. 5

E− embryos show enhanced depletion of many lyso-PL species during development when compared with E+ embryos. Data were generated and analyzed as described in Fig. 4. Notably, the lyso-PL species showing the greatest difference between the E− and E+ conditions all contained DHA (means±SEM). Two-way ANOVA, Bonferroni's post-test for multiple comparisons (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). LPC 16:0 and LPC 20:3 levels at 72 hpf were significantly altered in E− vs. E+ embryos when analyzed using unpaired t-tests and Sidak-Bonferroni post-test (p<0.001, higher in E−; and p=0.0176, lower in E−, respectively), as were LPI 20:4 levels at 120 hpf (p<0.0001, lower in E−).

Fig. 6

H₂¹⁸O incorporation into PC lipids. The CDP-choline pathway , is a representative metabolic pathway outlining reactions that allow for H₂¹⁸O incorporation into PCs and/or lyso-PCs. From the top, choline kinase (CK) phosphorylates choline to phosphocholine, which is converted to cytidine-diphosphocholine (CDP-choline) by CTP: phosphocholine cytidylyltransferase (CCT). Choline phosphotransferase (CPT) catalyzes PC synthesis. Fatty acyl chains are cleaved from the PC sn-2 position by phospholipase A₂ (PLA₂) to generate lyso-PC and free fatty acids. Free fatty acids are labeled (*), rapidly converted to acyl-CoA by acyl-CoA synthetase (ACS) and used for acylation of lyso-PL in a reaction catalyzed by acylglycerolphosphate acyltransferases (AGPAT). PC may be cleaved by PLC (phospholipase C) to yield diacylglycerides (DAG) and phosphocholine. Hydrolysis reactions in which incorporation of the ¹⁸O label may occur are shown in red, as are the indicated oxygens that may be labeled following PL remodeling.

Fig. 7

H₂¹⁸O incorporation into PCs and lyso-PCs is greater in E− than in E+ embryos. A and C. Ratios of [M+2+H]⁺/[M+H]⁺ from H₂O-incubated E− vs. E+ embryos were similar (solid bars; no significant differences) in PCs (A) and lyso-PCs (C); ratios comparing label incorporation in H₂¹⁸O-incubated E− vs. E+ embryo (patterned bars) revealed enhanced labeling in E− embryos of DHA-containing PCs, as well as in LPC 16:0 and 22:6, suggesting increased PL remodeling. PC species (means±SEM) were identified and confirmed as described in Fig. 4; PC 38:6 (16:0/22:6); PC 40:6 (18:0/22:6); PC 40:8 (18:2/22:6); PC 42:7 (20:3/22:4). All PLs and lyso-PLs showed significant (p<0.001) label incorporation in H₂O- vs. H₂¹⁸O-incubated embryos within the same diet condition, with the exception of E+ embryos LPC 22:6 in H₂O- vs. H₂¹⁸O-incubated (no significant difference, indicating no significant remodeling in E+ embryos). Asterisks indicate statistical differences between H₂¹⁸O E− and E+ groups (red and blue patterned bars, respectively). B and D. [M+2+H]⁺/[M+H]⁺ in H₂¹⁸O minus [M+2+H]⁺/[M+H]⁺ in H₂O (mean±SEM) in E− vs. E+ embryos for PCs (B) and lyso-PCs (D). A–D: Two-way ANOVA with Tukey's (A,C) or Sidak's (B,D) post-test for multiple comparisons (*p<0.05, ** p<0.01, ***p<0.001, ****p<0.0001).