Loss of the ceramide synthase HYL-2 from Caenorhabditis elegans impairs stress responses and alters sphingolipid composition

Abstract

Sphingolipids, essential membrane components and signaling molecules in cells, have ceramides at the core of their metabolic pathways. Initially termed as "longevity assurance genes", the encoding genes of ceramide synthases are closely associated with individual aging and stress responses, although the mechanisms remain unclear. This study aims to explore the alterations and underlying mechanisms of three ceramide synthases, HYL-1, HYL-2, and LAGR-1, in the aging and stress responses of Caenorhabditis elegans. Our results showed the knockdown of HYL-1 extends the lifespan and enhance stress resistance in worms, whereas the loss of HYL-2 function significantly impairs tolerances to heat, oxidation, and ultraviolet stress. Stress intolerance induced by HYL-2 deficiency may result from intracellular mitochondrial dysfunction, accumulation of reactive oxygen species, and abnormal nuclear translocation of DAF-16 under stress conditions. Loss of HYL-2 led to a significant reduction of predominant ceramides (d17:1/C20∼C23) as well as corresponding complex sphingolipids. Furthermore, the N-acyl chain length composition of sphingolipids underwent dramatic modifications, characterized by a decrease in C22 sphingolipids and an increase in C24 sphingolipids. Extra d18:1-ceramides resulted in diminished stress resilience in wild-type worms, while supplementation of d18:1/C16 ceramide to HYL-2-deficient worms marginally improved stress tolerance to heat and oxidation. These findings indicate the importance of appropriate ceramide content and composition in maintaining subcellular homeostasis and nuclear-cytoplasmic signal transduction during healthy aging and stress responses.

Keywords: Caenorhabditis elegans; ROS; ceramide synthases; sphingolipids; stress tolerance.

Figure 1

HYL-2 mutant reduces normal life history traits and stress tolerances in C. elegans.A, survival curves of wild-type (N2, n = 123) and ceramide synthase mutant animals, hyl-1(ok976, n = 145), hyl-2(ok1766, n = 144), and lagr-1(gk331, n = 150), under standard culture conditions were plotted and analyzed. Days were counted from egg lay as day 0 for all animals (abscissa). B, images of WT and ceramide synthase mutation animals at the day 4 stage captured using a Nikon optical microscope. Scale bar, 1 mm. C, the body lengths of day 4 animals were measured under a microscope and statical analyzed. n > 200. D, the duration from a fresh egg (F1) to the first egg (F2) laid by each hatched nematode (F1) was monitored under normal culture conditions. E, day 7 nematodes were placed in M9 buffer and counted for their total body bends in 1 min. Results are presented as mean values. The p values are relative to WT (N2) animals analyzed by two-tailed student’s t test (∗∗p < 0.01; ∗∗∗p < 0.001). F, day 7-stage wild-type (N2, n = 66), hyl-1(ok976, n = 73), hyl-2(ok1766, n = 76), and lagr-1(gk331, n = 75) nematodes were subjected to survival analysis in a 35 °C incubator. G, survival of Day 7-stage wild-type (N2, n = 143), hyl-1(ok976, n = 166), hyl-2(ok1766, n = 98), and lagr-1(gk331, n = 113) nematodes were monitored under oxidative stress induced by 240 μM juglone. H, the survival of wild-type (N2, n = 50), hyl-1(ok976, n = 50), hyl-2(ok1766, n = 47), and lagr-1(gk331, n = 49) animals was assessed after exposure to four rounds of UV radiation from the day 7 stage.

Figure 2

Sphingolipidomic analysis reveals the significant alteration in sphingolipid composition caused by deletion of hyl-2.A, partial least squares discriminant analysis (PLS-DA) of quantified SLs shows separation of N2 worms with the three CerS mutant strains. B–F, heatmap showing log2-fold change in the amount of sphingolipids species in the mutant strains compared with N2 worms (n = 3 per group). The white color is set as the average of all samples, with increasing red indicating higher lipid levels and increasing blue indicating lower levels. B, analysis of total levels of Cer, HexCer, and SM reveals a decrease in sphingolipid abundance in the posterior averages of hyl-1, hyl-2, and lagr-1 knockouts with hyl-2 deficiency showing the most pronounced reduction. C, decrease in the levels of atypical sphingolipids paticularly a significant reduction in C22 1-deoxyDHceramide in hyl-2 deficiency. D–F, relative levels of Cer, HexCer, and SM with different N-acyl chain lengths. D, hyl-1, hyl-2, and lagr-1 gene knockout samples show decreased ceramide levels, with hyl-1 mutant exhibiting a significant reduction in ceramides with C16 to 18, ≥C24 acyl chain lengths. The hyl-2 mutant demonstrated a pronounced decrease in ceramide levels with C20∼23 acyl chain lengths, while lagr-1 mutant displayed a non-specific reduction in ceramide levels across various chain lengths. E and F, HexCer and SM also exhibit variations in levels dependent on the N-acyl chain length, similar to the dependency observed in ceramide. The relative levels of sphingomyelins were analyzed, revealing differences in abundance based on the length of the acyl chains. G, the percentage of subtypes of sphingolipids based on N-acyl chain length classification in sphingolipidomics.

Figure 3

Hyl-2 is required for maintaining ROS homeostasis under heat and oxidative stress.A, wild-type (N2) and ceramide synthase mutant nematodes were exposed to 35 °C heat or 240 μM juglone-induced oxidative stress. Intracellular ROS levels of worms were assessed by monitoring green fluorescence using dichlorofluorescein (DCF). Scale bar, 100 μm. B, mitochondrial ROS levels were determined by staining with MitoTracker Red CMXRos (MTRC), represented by red fluorescence. Scale bar, 100 μm. The rightmost graphs display the quantitative results for each condition. Error bars represent SD (two-tailed student’s t test, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). C, fluorescence micrographs showed MitoTracker Deep Red (MTDR)-labeled mitochondria in wild-type (N2) and mutant nematodes, with a higher magnification image of the mitochondria. D, compared to wild-type worms (N2), hyl-2(ok1766) worms exhibited a lower mitochondrial copy number at day 7 of adulthood. E, ATP production was reduced in hyl-2(ok1766) worms at day 7 of adulthood compared to wild-type worms (N2). Scale bar of main image panel, 100 μm and associated zoom magnification image panel, 10 μm. Error bars represent SD (n = 3, two-tailed student’s t test, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). F and G, comparison of body wall muscle mitochondrial between hyl-2(RNAi)-treated and Control(RNAi)-treated SJ4103 strain worms at day 7 under 35 °C heat or 240 μM juglone-induced oxidation stress, using a mitochondrially localized GFP reporter. The white arrow indicates abnormal mitochondrial morphology, Scale bar, 10 μm.

Figure 4

Functional loss of hyl-2 or lagr-1 disturbs DAF-16 nuclear accumulation in response to stress.A, mild heat stress (30 °C, 8 h) induced differential nuclear accumulation of DAF-16 in response to either empty vector control bacteria Control(RNAi) or the indicated RNAi strains. hyl-2(RNAi) and lagr-1(RNAi) led to reduced nuclear entry of DAF-16 under heat stress, while hyl-1(RNAi) showed similar effects to control bacteria. Scale bar of main image panel, 100 μm and associated zoom magnification image panel, 40 μm. B, quantitative RT-PCR (qRT-PCR) analysis of sod-3 expression in wild-type (N2) and mutant nematodes at day 7 after heat stress (30 °C, 10 h) and oxidative stress (120 μM juglone, 10 h). Error bars represent SD (n = 3, two-tailed student’s t test, ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). C–E, cumulative survival curves of N2 and daf-16(mu86) under 35 °C heat stress, exposed to either empty vector control bacteria Control(RNAi) or the indicated RNAi strains. The heat stress response of the indicated RNAi strains mirrored the corresponding mutant lines. C, when subjected to hyl-1(RNAi), the reduced lifespan of daf-16(mu86) was extended but not fully restored to the level of N2 animals. D and E, when subjected to hyl-2(RNAi) and lagr-1(RNAi), the shortened lifespan of N2 did not appear in daf-16(mu86) animal.

Figure 5

Exogenous supplementation of C16 ceramide marginally rescues the decrease in stress tolerance caused by HYL-2 dysfunction.A and B, supplementation of d18:1/C16 ceramide in HYL-2-deficient worms marginally rescued their stress tolerance and the expression of mitochondrial fission and fusion genes (C). D, this supplementation did not lead to further accumulation of reactive oxygen species (ROS).