Impaired embryonic development in glucose-6-phosphate dehydrogenase-deficient Caenorhabditis elegans due to abnormal redox homeostasis induced activation of calcium-independent phospholipase and alteration of glycerophospholipid metabolism

Abstract

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a commonly pervasive inherited disease in many parts of the world. The complete lack of G6PD activity in a mouse model causes embryonic lethality. The G6PD-deficient Caenorhabditis elegans model also shows embryonic death as indicated by a severe hatching defect. Although increased oxidative stress has been implicated in both cases as the underlying cause, the exact mechanism has not been clearly delineated. In this study with C. elegans, membrane-associated defects, including enhanced permeability, defective polarity and cytokinesis, were found in G6PD-deficient embryos. The membrane-associated abnormalities were accompanied by impaired eggshell structure as evidenced by a transmission electron microscopic study. Such loss of membrane structural integrity was associated with abnormal lipid composition as lipidomic analysis revealed that lysoglycerophospholipids were significantly increased in G6PD-deficient embryos. Abnormal glycerophospholipid metabolism leading to defective embryonic development could be attributed to the increased activity of calcium-independent phospholipase A₂ (iPLA) in G6PD-deficient embryos. This notion is further supported by the fact that the suppression of multiple iPLAs by genetic manipulation partially rescued the embryonic defects in G6PD-deficient embryos. In addition, G6PD deficiency induced disruption of redox balance as manifested by diminished NADPH and elevated lipid peroxidation in embryos. Taken together, disrupted lipid metabolism due to abnormal redox homeostasis is a major factor contributing to abnormal embryonic development in G6PD-deficient C. elegans.

Figure 1

The impact of G6PD deficiency on C. elegans embryo physiology. (a) The effect of G6PD knockdown on C. elegans embryo morphology under physiological condition (Egg buffer) and osmotic stress (Water, KCl). Representative DIC images of embryos derived from Mock and G6PD(RNAi)(Gi) C. elegans are shown. White scale bar indicates 10 μm. (b) The effect of G6PD knockdown and fatty acid synthase knockdown (Fasn-1(RNAi)) on permeability barrier formation in C. elegans embryos. Representative DIC and merged fluorescent images of two-cell embryos derived from Mock, Gi and Fasn-1(RNAi) of OD344 C. elegans are shown. In Mock embryo, the permeability barrier prevented the diffusion of red fluorescence (mCherry::CPG-2) to the surface of embryos (white arrow). The permeability barrier was disrupted in Gi embryos. Such inhibition caused the mCherry::CPG-2 to fill the space between the eggshell and embryo surface (white arrow head). Green fluorescence indicates the plasma membrane of the embryo. White scale bar indicates 10 μm. (c) List of dye permeability ratio in Mock and Gi embryos with different dyes (n>100 embryos/group)

Figure 2

G6PD deficiency disrupts eggshell structure in C. elegans embryo indicated by TEM. (a) Schematic drawing of a normal C. elegans embryo. Red box indicates a portion of eggshell structure shown in (b). (b) Color-coded cartoon of the normal eggshell structure shown in (c). (c) Representative TEM image of the Mock embryo eggshell structure. (d) Representative TEM image of G6PD-deficient embryo eggshell structure. Black scale bar in (c and d) indicates 0.2 μm

Figure 3

G6PD deficiency substantially increases lysoglycerophospholipids in C. elegans embryo as revealed by lipidomic analysis. (a) Workflow of lipidomic analysis. (b) The PCA plots of Mock, Gi and Fasn-1(RNAi) embryos in ESI⁺ and ESI⁻ mode. (c) Comparison of lysoglycerophospholipids between Mock and Gi embryos in ESI⁺ mode. (d) Comparison of lysoglycerophospholipids between Mock and Gi adults in ESI⁺ mode. All fold change data are presented as the mean±S.D. and statistical significance was calculated using a two-tailed t-test (N=3, *P<0.05; **P<0.005; ***P<0.001)

Figure 4

iPLA activity in G6PD deficiency-induced embryonic lethality. (a) The iPLA activity was determined in Mock and Gi adults (N=3, *P<0.05) and embryos (N=7, **P<0.005)(^##P<0.005, Gi embryos compared with Gi adults). (b) Effect of iPLA inhibition on brood size (N>3, n>60 worms/experiment, *P<0.05; **P<0.005; ***P<0.001)

Figure 5

G6PD deficiency disrupts redox homeostasis by enhancing lipid peroxidation and reducing NADPH production. (a) Lipid peroxidation was determined in Mock and Gi embryos (N=4, *P<0.05). (b) NADPH and NADP levels were determined in Mock and Gi adults (N>3, *P<0.05)

Figure 6

Proposed model of how G6PD deficiency causes embryonic lethality in C. elegans