Sphingolipid biosynthetic inhibitor L-Cycloserine prevents oxidative-stress-mediated death in an in vitro model of photoreceptor-derived 661W cells

Abstract

Oxidative stress plays a pivotal role in the pathogenesis of several neurodegenerative diseases. Retinal degeneration causes irreversible death of photoreceptor cells, ultimately leading to vision loss. Under oxidative stress, the synthesis of bioactive sphingolipid ceramide increases, triggering apoptosis in photoreceptor cells and leading to their death. This study investigates the effect of L-Cycloserine, a small molecule inhibitor of ceramide biosynthesis, on sphingolipid metabolism and the protection of photoreceptor-derived 661W cells from oxidative stress. The results demonstrate that treatment with L-Cycloserine, an inhibitor of Serine palmitoyl transferase (SPT), markedly decreases bioactive ceramide and associated sphingolipids in 661W cells. A nontoxic dose of L-Cycloserine can provide substantial protection of 661W cells against H₂O₂-induced oxidative stress by reversing the increase in ceramide level observed under oxidative stress conditions. Analysis of various antioxidant, apoptotic and sphingolipid pathway genes and proteins also confirms the ability of L-Cycloserine to modulate these pathways. Our findings elucidate the generation of sphingolipid mediators of cell death in retinal cells under oxidative stress and the potential of L-Cycloserine as a therapeutic candidate for targeting ceramide-induced degenerative diseases by inhibiting SPT. The promising therapeutic prospect identified in our findings lays the groundwork for further validation in in-vivo and preclinical models of retinal degeneration.

Keywords: Ceramide biosynthesis; L-Cycloserine; Oxidative stress; Photoreceptors; Retina; Retinal cell death; Serine palmitoyl transferase (SPT); Sphingolipids; Therapeutic intervention.

Figure 1:. L-Cs reduce ceramide and other sphingolipids in 661W cells.

Analysis of total sphingolipid levels in 661W cells (n = 5/group) treated with different L-Cs doses (10–50 μM) for 24 hours showing levels of total ceramide (Cer), monohexosylceramide (MHC), sphingomyelin (SM) and total sphingolipid (SPL). (Values are represented as mean ± SEM; t-test; *p < 0.05, **p < 0.01, ***p < 0.001). Control: cells having no treatment with L-Cs; L-Cs10: treated with 10 μM L-Cs for 24 h; L-Cs25: treated with 25 μM L-Cs for 24 h; L-Cs50: treated with 50 μM L-Cs for 24 h.

Figure 2A:. Cell viability assay for 661W cells using L-Cs.

661W cells were treated with different doses of L-Cs (0–400 μM) for 24 hours. Cell viability was then determined by thiazolyl blue (MTT) assay (n=3 plate × 3 replication assay). Values are represented as mean ± SEM; One-way ANOVA; *p<0.05, **p < 0.01). Figure 2B: Cell viability assay for 661W cells using L-Cs. 661W cells were treated with different doses (10 and 25 μM) of L-Cs for 24 hours. Cell death was then measured by analyzing the release of lactate dehydrogenase (LDH; n=4 plate × 4 replication assay). Values are represented as mean ± SEM; t-test; **p < 0.01. Figure 2C: Cell viability assay for 661W cells using L-Cs. 661W cells were treated with different doses (5,10, 25 and 50 μM) of L-Cs for 3 hours. Cell death was then measured by analyzing the release of lactate dehydrogenase (LDH; n=4 plate × 4 replication assay). Values are represented as mean ± SEM; no statistical significance was found.

Figure 3A:. Dose dependent cytotoxicity of H₂O₂.

Treatment of 661W cells with different doses of H₂O₂ (100–750 μM) for 3 hours; 10% Triton was used as positive control. Cell death was then measured by analyzing the release of lactate dehydrogenase (LDH; n=4 plate × 3 replication assay). Figure 3B: L-Cs protects 661W cells from oxidant-induced cell death. 661W cells were pretreated with different doses (10 and 25 μM) of L-Cs for 24 hours followed by 300 μM H₂O₂ for 3 hours. Cell death was then measured by analyzing the release of lactate dehydrogenase (LDH; n=4 plate × 4 replication assay). Values are represented as mean ± SEM; t-test; **p < 0.01. Figure 3C: L-Cs protects 661W cells from oxidant-induced cell death. 661W cells were cotreated with 300 μM H₂O₂ and different doses of L-Cs (5,10, 25 and 50 μM) for 3 h. Cell death was then measured by analyzing the release of lactate dehydrogenase (LDH; n=4 plate × 4 replication assay). (Values are represented as mean ± SEM; t-test; *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 4A:. L-Cs treatment alters level of ceramide and other sphingolipids in H₂O₂ induced oxidative stressed 661W cells.

Analysis of total fatty acid composition of ceramide (Cer), sphingomyelin (SM), monohexosylceramide (MHC) and all sphingolipids (SPL) in 661W cells (n = 6/group). Control: cells having no treatment with L-Cs or H₂O₂; L-Cs: treated with 10 μM L-Cs for 3 h; H₂O₂: treated with 300 μM of H₂O₂ for 3 h; L-Cs + H₂O₂: cotreated with 10 μM L-Cs and 300 μM H₂O₂ for 3 h. Values are presented as mean ± SEM; t-test; # represents significance between H₂O₂ and L-Cs + H₂O₂ treatment; #p < 0.05. Figure 4B: L-Cs treatment alters ceramide and other sphingolipids in H₂O₂ induced oxidative stressed cells. Analysis of major sphingolipid levels represented in a pie chart by absolute value (pmol/ mg of protein) showing total composition of ceramide (Cer), sphingomyelin (SM) and monohexosylceramide (MHC) percent in 661W cells (n = 6/group). B1 represents control or cells having no treatment with L-Cs or H₂O₂; B2 represents cells treated with 300 μM of H₂O₂ for 3 h; B3 represents cells treated with 10 μM L-Cs for 3 h; B4 represents cells cotreated with 10 μM L-Cs and 300 μM H₂O₂ for 3 h.

Figure 5:. Gene expression in 661W cells measured by quantitative reverse transcriptase PCR (qRT–PCR).

Gene expression was analyzed with the comparative Cq value method after normalizing against the housekeeping genes, Gapdh and Rpl19. Expression values (±SEM) are presented against fold change over control value, which was set to 1.0 (n=3 samples ×3 replication assay per sample). Control: cells having no treatment with L-Cs or H₂O₂; L-Cs: treated with 10 μM L-Cs for 3 h; H₂O₂: treated with 300 μM of H₂O₂ for 3 h; L-Cs + H₂O₂: cotreated with 10 μM L-Cs and 300 μM H₂O₂ for 3 h (* represents significance between control and H₂O₂; *p < 0.05, **p < 0.01; ***p < 0.001, by the student t test; $ represents significance between H₂O₂ and L-Cs + H₂O₂ treatment; $p < 0.05, $ $p < 0.01, $ $ $p < 0.001, by the student t test; unmarked bars implies no significance between the comparative groups).

Figure 6:. Expression and quantification of selected proteins in 661W cells cotreated with L-Cs and H₂O₂.

A: Expression and quantification of heme oxygenase 1 (Ho1) and Poly (ADP-ribose) polymerase (PARP) proteins in 661W cells was measured by western blot analysis. Proteins were extracted and subjected to western blot with anti-Ho1 and anti-PARP antibodies. Lane 1 (control): no treatment; lane 2 (L-Cs): 10 μM L-Cs treated cells; lanes 3 and 4 (H₂O₂): 300 μM H₂O₂ treated cells; lane 5 (L-Cs+ H₂O₂): cotreated with 10 μM L-Cs and 300 μM H₂O₂. B: Quantification of Ho1 in 661W cells with western blot. C: Quantification of PARP in 661W cells with western blot. Quantification of Ho1 and PARP was obtained with densitometric analysis and normalized with β-actin. (n=3; values are represented as mean ± SEM; t-test; *p < 0.05, ***p < 0.001).