Acid ceramidase modulates the lipid profile and exacerbates sensitivity to ferroptosis in senescent cells

Abstract

Cellular senescence, a complex biological process characterized by irreversible cell cycle arrest and the senescence-associated secretory phenotype, has emerged as a critical target for therapeutic development for age-related diseases. Ferroptosis, an iron-dependent regulated cell death pathway driven by the accumulation of lipid peroxidation in cell membranes, has been implicated in neurodegenerative diseases and other age-related disorders. This study investigated the relationship between cellular senescence and ferroptosis. Using human fetal lung Wi-38 fibroblasts induced to senesce via replicative exhaustion, we report a novel role for acid ceramidase (ACase), which breaks down ceramides into sphingosine and free fatty acids, in regulating the sensitivity of senescent cells to lipid peroxidation and ferroptosis through the modulation of polyunsaturated fatty acid composition of membrane phospholipids. Furthermore, we demonstrate a cell non-autonomous paracrine sensitization of non-senescent cells to ferroptosis by senescent cells. Together, these findings unveil ACase as a novel regulator of the ferroptosis pathway and open promising therapeutic avenues for targeting senescence-linked disorders and advancing healthy aging strategies.

Figure 1.. Senescent cells are more sensitive to ferroptosis.

(A) Percentage of survival of cell populations of increasing PDL and senescent (sen.) cells after RSL3 (250nM) treatment. (B) Percentage of cell survival of senescent cells after RSL3 (250nM) and/or J147 (1μM) treatment. (**p < 0.01, ****p < 0.0001. One-way ANOVA) Representative flow cytometry histograms (C) and quantification (D) showing LPO levels (C11-Bodipy 581/591) in proliferative (prol.) and senescent (sen.) cells in the presence or absence of RSL3 (250nM). (**p < 0.01, ***p < 0.001, ****p < 0.0001. Two-way ANOVA). Values represent the mean ± SEM of at least 3 independent experiments. LPO, lipid peroxidation.

Figure 2.. Acid ceramidase knock-down protects proliferative and senescent cells against ferroptosis independently of GSH.

(A) Percentage of cell survival of senescent cells after RSL3 treatment in the presence or absence of siACSL4 or siACase (B) Representative blot of acid ceramidase protein expression in proliferative cells of increasing PDL and senescent cells. (C) Representative blot showing acid ceramidase knock-down after siRNA transfection (siACase). (D) Representative blot of p21 protein expression in the presence or absence of siACase. (E) Representative micrographs showing β-galactosidase activity (blue staining). (F) Quantification of secreted interleukin-6 and interleukin-8 levels measured by ELISA. (G) Percentage of cell survival of proliferative and senescent cells after RSL3 treatment in the presence or absence siACase. (H) Representative flow cytometry histograms and quantification (I) showing LPO levels (C11-Bodipy 581/591) in proliferative and senescent cells in the presence or absence of RSL3 (250nM) and siACase. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Two-way ANOVA). Values represent the mean ± SEM of at least 3 independent experiments. PDL, population doubling level; siC, control siRNA; siA, ACase siRNA; LPO, lipid peroxidation.

Figure 3.. Acid ceramidase knock-down decreases lipid peroxidation independently of Fe²⁺ regulation.

(A) Quantification of GSH levels in proliferative and senescent cells after the indicated treatments. (B) Percentage of cell survival of proliferative and senescent cells after GSH depletion by BSO treatment in the presence or absence siACase. Co-treatment with RSL3 is indicated (200nM). (C) Representative blot and quantification (bar graphs) of GPX4 protein expression in proliferative and senescent cells in the presence or absence of siACase. (D) Representative blot and quantification (bar graphs) of FTH1 protein expression in proliferative and senescent cells in the presence or absence of siACase. (E) Representative flow cytometry histograms and quantification (bars graph) showing labile iron pool levels (FerroFarRed) in proliferative and senescent cells in the presence or absence of siACase. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Two-way ANOVA). Values represent the mean ± SEM of at least 3 independent experiments. GSH, glutathione; LIP, labile iron pool.

Figure 4.. Senescent cells show a general increase in PUFA-containing phospholipids compared to proliferative cells and this is countered by acid ceramidase inhibition.

(A) Diagram representing the enzymatic relation between Cer, SM, sphingosine and FAs. (B) Relative fold change of ceramide and sphingomyelin in the indicated conditions. (C) Relative fold change of the most abundant (≥ 80% of total) ^sn-2PUFA organized by PL class and ordered by abundance inside each PL class (%) from top to bottom. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. One-way ANOVA). (D) Representative blot and quantification (bar graphs) of ACSL4 protein expression., (**p < 0.01, ***p < 0.001, ****p < 0.0001. Two-way ANOVA). Values represent the mean ± SEM of at least 3 independent experiments. Cer, ceramide; SM, sphingomyelin; SFA/MUFA, saturated fatty acids/monounsaturated fatty acids; CerS, ceramide synthase; SMS, sphingomyelin synthase; prol, proliferative; sen, senescent; siC, CTRL siRNA; siA, ACase siRNA; AA, arachidonic acid; DHA, docosahexaenoic acid; DPA, docosapentanoic acid; DGLA, dihomo-gamma-linoleic acid; LA, linoleic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; PG, phosphatidylglycerol.

Figure 5.. Paracrine signaling from senescent cells increases ACase expression and ferroptosis sensitivity in proliferative cells.

(A) Representative blot and quantification (bar graphs) of ACase and ACSL4 protein expression of proliferative cells incubated with IL-6/8 (50/25 ng/ml) for the indicated times. (*p < 0.05, ***p < 0.001. Two-way ANOVA). (B) Percentage of cell survival of proliferative cells after RSL3 treatment (250nM) in the presence or absence of IL-6/8 (72 h). (C) Representative flow cytometry histograms and quantification (bar graphs) showing LPO levels (C11-Bodipy 581/591) in proliferative cells after RSL3 treatment (250nM) in the presence or absence of IL-6/8, (**p < 0.01, ***p < 0.001, ****p < 0.0001. Two-way ANOVA). Values represent the mean ± SEM of at least 3 independent experiments. sen, senescent; LPO, lipid peroxidation.

Figure 6.. Working model illustrating the possible interplay between ACase and ferroptosis in senescent cells.

The upregulation of ACase in senescent cells would increase the pool of SFA/MUFA from the Cer breakdown (1). Through the Kennedy pathway (2) and/or the Lands cycle (3), increased ^sn-2SFA/PUFA-PL would lead to an accumulation of ^sn-2PUFA-PL, priming the cell membranes for LPO and ferroptosis (4). SM, sphingomyelin; Cer, ceramide; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyunsaturated fatty acid; PL, phospholipid; ROS, reactive oxygen species; LPO, lipid peroxidation; GSH, reduced glutathione; GSSG, oxidized glutathione.