Mitochondrial metabolism and epigenetic crosstalk drive the SASP

Abstract

Senescent cells drive tissue dysfunction through the senescence-associated secretory phenotype (SASP). We uncovered a central role for mitochondria in the epigenetic regulation of the SASP, where mitochondrial-derived metabolites, specifically citrate and acetyl-CoA, fuel histone acetylation at SASP gene loci, promoting their expression. We identified the mitochondrial citrate carrier (SLC25A1) and ATP-citrate lyase (ACLY) as critical for this process. Inhibiting these pathways selectively suppresses SASP without affecting cell cycle arrest, highlighting their potential as therapeutic targets for age-related inflammation. Notably, SLC25A1 inhibition reduces systemic inflammation and extends healthspan in aged mice, establishing mitochondrial metabolism as pivotal to the epigenetic control of aging.

Figure 1. Mitochondria modulate histone acetylation at SASP loci.

(a) Schematic representation of the experimental setup. (b) Western blot analysis of mitochondrial proteins UQCRC2 and NDUFB8, confirming the complete loss of mitochondria in IMR90 Parkin senescent cells (Sen) following CCCP treatment. c. Metaplot and heatmap analysis of SASP genes showing decreased histone acetylation (H3K27ac) in senescent cells lacking mitochondria (Sen+CCCP). Top: Metaplot depicting a composite sum of all normalized H3K27ac enrichment at SASP gene signals; Bottom: heatmap of H3K27ac enrichment normalized by total H3 (±5kb from TSS). Data are averaged from n=3 independent experiments. (d-e). Browser track (left panel) and mRNA expression (right panel) of IL6 (d) and CCL2 (e) genes displaying H3K27ac distribution. qPCR data, n=4. (f). Scheme of the experimental workflow. (g). Representative immunofluorescence images of nuclear acetylated lysine (green) and mitochondria staining (TOMM20, red). Scale bar 50μm. (h). Quanti cation of nuclear acetyllysine per cell from n=3 independent experiments. (i). mRNA expression levels of various SASP genes (n=3 per condition). Data are expressed as Mean ± S.E.M. One way ANOVA or Student’s t-test *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

Figure 2. Upregulation of mitochondrial pyruvate carriers (MPC) in senescence modulates the SASP.

(a) Schematic illustration of MPC localization and function within the mitochondria. (b) Western blot analysis showing increased MPC1 protein levels in different models of senescence: irradiation-induced (Sen(IR)), doxorubicin-induced (Sen(Dox)), and replicative senescence (Sen(RS)). (c) mRNA expression levels of MPC1 and MPC2 across senescence models. (d) Mass spectrometry quantification of TCA cycle metabolites in senescent cells following treatment with UK5099 (MPC inhibitor) (n=3 independent experiments). (e) Column-clustered heatmap showing differential expression of SASP genes in senescent cells, with downregulation following UK5099 treatment. Color intensity represents column Zscores (red: high expression, blue: low expression). (f) Cytokine array heatmap of 24-hour conditioned media from senescent cells, showing cytokine expression changes. (g) Column-clustered heatmap of cell cycle-associated genes in senescent cells, which are not affected by UK5099 treatment (color intensity as in panel e). (h) Western blot indicating that UK5099 does not reduce expression of p16 and p21, nor restore Cyclin A levels, confirming a lack of effect on cell cycle arrest. For RNA-seq and cytokine arrays, n=3 independent conditions were analyzed. Data are presented as Mean ± S.E.M. Statistical tests: Student’s t-test for (c) and one-way ANOVA for (d), *p<0.05; **p<0.01; ***p<0.001; ***p<0.0001.

Figure 3. Upregulation of the Mitochondrial Citrate Carrier (SLC25A1) modulates the SASP in senescent cells.

(a) Schematic illustration of SLC25A1 localization and function within mitochondria. (b) Western blot showing increased SLC25A1 protein levels in senescence models: irradiation-induced (Sen(IR)), doxorubicin-induced (Sen(Dox)), and replicative senescence (Sen(RS)). (c) mRNA expression of SLC25A1 in these senescence models compared to proliferative cells. (d) Western blot confirming successful CRISPR/Cas9-mediated deletion of SLC25A1 in proliferative (Prol) and senescent (Sen(IR)) cells, with persistent p16 expression in senescent cells after knockout. (e-g) mRNA expression of cyclin-dependent kinase inhibitors (cdki) in different senescence models. (h-n) mRNA expression of various SASP components under the same conditions. All values are presented as fold change relative to proliferative cells (Prol) (n=6–9 per condition). Data are shown as Mean ± S.E.M. *One-way ANOVA test: *p<0.05; **p<0.01; ***p<0.001; ***p<0.0001.

Figure 4. Pharmacological inhibition of Mitochondrial Citrate Carrier (SLC25A1) with CTPI2 reduces the SASP in senescent cells.

(a) Schematic illustrating experimental approach. (b-g) mRNA expression levels of SASP components (b-e) and cyclin-dependent kinase inhibitors (cdki) (f-g) in senescent cells (Sen(IR)) treated with two doses of CTPI2 (15 and 30 μM), expressed as fold change relative to proliferative cells (Prol) (n=6–12). (h) Western blot analysis showing that CTPI2 treatment does not alter p16 or p21 expression, nor does it restore Cyclin A levels, indicating that the cell cycle arrest is maintained. (i) Gene ontology pathways of genes downregulated by CTPI2 compared to untreated senescent cells. (j-k) Column-clustered heatmaps showing SASP factors (j) and cell cycle-associated genes (k) differentially expressed in senescent cells and downregulated by CTPI2 treatment (color intensity represents column Z-scores, where red indicates high expression and blue indicates low expression; n=3 independent experiments). (l) Representative immunofluorescence images of nuclear acetylated lysine (green) in two models of senescence treated with or without CTPI2. Scale bar = 50 μm. (m) Quantification of nuclear acetyl-L-lysine integrated density per cell (n=3 independent experiments). (n) Western blot showing reduced histone acetylation (H3K27ac) after CTPI2 treatment across two different senescence inducers. (o) Metaplot (top) and heatmap (bottom) of SASP genes showing reduced histone H3K27 acetylation (H3K27ac) levels normalized by histone H3 around the transcription start sites (±5 kb) in senescent cells (Sen(IR)) after CTPI2 treatment (data averaged from n=3 independent experiments). (p) Browser tracks of IL6 and CCL2 genes depicting reduced H3K27ac levels following CTPI2 treatment. Data are shown as Mean ± S.E.M. *One-way ANOVA or Student’s t-test: *p<0.05; **p<0.01; ***p<0.001; ***p<0.0001.

Figure 5. Mitochondria-derived Citrate drives the SASP through ACLY activity.

(a) Schematic representation illustrating the export of citrate from mitochondria and conversion to Acetyl- CoA. (b) Relative mRNA expression of SASP components in proliferative cells treated with 10 mM citrate (Citrate) compared to untreated proliferative cells (Control), shown as fold change. (c) Western blot analysis of histone H3 acetylation (H3K27ac) and total histone H3, demonstrating increased histone acetylation in citrate-treated cells. (d) Relative mRNA expression of ACLY in cells undergoing senescence induced by various stimuli. (e) mRNA expression in irradiation-induced senescence (Sen(IR)) showing successful ACLY knockdown by siRNA and subsequent effects on SASP gene components, expressed as fold change relative to proliferative cells (Prol). (f) Western blot analysis showing reduced histone H3 acetylation (H3K27ac) following ACLY silencing in Sen(IR) cells. (g) mRNA expression in replicative senescence (Sen(RS)) showing effective ACLY knockdown by siRNA and effects on SASP gene expression, presented as fold change relative to proliferative cells (Prol). (h) Western blot analysis indicating decreased histone H3 acetylation (H3K27ac) in Sen(RS) cells following ACLY silencing. Data are represented as Mean ± S.E.M. Statistical analysis was performed using Student’s t-test for (b, d) and one-way ANOVA for (e, g). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 6. Pharmacological inhibition of Mitochondrial Citrate Carrier (SLC25A1) during aging improves frailty and muscle function.

(a) Schematic representation of the in vivo experimental procedure detailing CTPI2 treatment in aged mice. (b) Representative images of male and female mice treated with either vehicle or CTPI2, as indicated by the green arrows. (c) Frailty index scores at baseline (0 months) and after 3 months of treatment (final) in vehicle (n = 19) and CTPI2-treated (n = 19) mice (males and females combined). (d) Histogram of frailty index scores after 3 months of treatment, showing individual values for vehicle (n = 19) and CTPI2-treated (n = 19) mice. Males are denoted by filled black dots, and females by open black dots. (e) Forelimb grip strength measured at baseline, 1.5 months, and 3 months (final) in each group (n = 19 per group). (f) Representative images of WGA staining, marking the membrane of cross-sectional myofibers. (g) Quantification of mean cross-sectional myofiber area per mouse. (h) Distribution of cross-sectional myofiber area per mouse. (i) Percentage of centrally nucleated fibers per mouse, presented separately for females (open dots, left panel) and males (filled dots, right panel). (j-k) mRNA expression of SASP components (j) and cell cycle inhibitors (p16, p21) (k) in quadriceps muscle from male and female mice, expressed as fold change relative to vehicle-treated mice. (l) Correlation curves of Il1b or Il6 expression with forelimb grip strength for each mouse. Data are presented as Mean ± S.E.M. Statistical analysis performed using Student’s t-test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Vehicle: n = 10 males and 9 females; CTPI2: n = 9 males and 10 females. Males are represented by filled dots, and females by open dots.