Calcium channel ITPR2 and mitochondria-ER contacts promote cellular senescence and aging

Abstract

Cellular senescence is induced by stresses and results in a stable proliferation arrest accompanied by a pro-inflammatory secretome. Senescent cells accumulate during aging, promoting various age-related pathologies and limiting lifespan. The endoplasmic reticulum (ER) inositol 1,4,5-trisphosphate receptor, type 2 (ITPR2) calcium-release channel and calcium fluxes from the ER to the mitochondria are drivers of senescence in human cells. Here we show that Itpr2 knockout (KO) mice display improved aging such as increased lifespan, a better response to metabolic stress, less immunosenescence, as well as less liver steatosis and fibrosis. Cellular senescence, which is known to promote these alterations, is decreased in Itpr2 KO mice and Itpr2 KO embryo-derived cells. Interestingly, ablation of ITPR2 in vivo and in vitro decreases the number of contacts between the mitochondria and the ER and their forced contacts induce premature senescence. These findings shed light on the role of contacts and facilitated exchanges between the ER and the mitochondria through ITPR2 in regulating senescence and aging.

Fig. 1. Itpr2 knockout increases lifespan and limits age-related phenotypes in mice.

a Survival curves of Itpr2 WT (n = 9) or KO (n = 11) C57BL/6 female mice. Log-rank test. b Immunophenotyping of the spleens of 23-month-old mice displaying a relative number of memory (CD44high) CD4+ and CD8+ T-cells in Itpr2 WT (n = 13) and KO (n = 10) female and male mice. Mean ± SEM. Unpaired two-tailed Student t-test. c Micrographs of liver sections stained with Sirius Red of 23-month-old male and female Itpr2 WT (n = 12) and KO (n = 10) mice. Scale bar: 25 µm. Quantification of the percentage of mice presenting macroscopic steatosis. d mRNA level extracted from microarray data in the livers of 23-month-old WT (n = 4) and KO (n = 4) female mice. Mean ± SEM. Unpaired two-tailed Student t-test. e Micrographs of liver sections stained with Sirius Red. Scale bar: 10 µm. Quantification of relative intrahepatic collagen fibers according to Sirius Red staining in 23-month-old male and female Itpr2 WT (n = 12) and KO (n = 10) mice. Mean ± SEM. Two-tailed Mann–Whitney U Test. f Quantification of relative blood AST level of 23-month-old Itpr2 WT (n = 14) and KO (n = 13) female and male mice. Mean ± SEM. Unpaired two-tailed Student t-test. g Intraperitoneal glucose-tolerance test of adult (20-month-old) and old (26-month-old) Itpr2 WT (n = 6) and KO (n = 8) male mice. Mean ± SEM. Unpaired two-tailed Mann–Whitney U Test. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 2. Loss of Itpr2 in mice reduces key features of cellular senescence.

a Inflammatory response gene ontology obtained from the genes downregulated in the livers of 23-month-old Itpr2 WT (n = 4) and KO (n = 4) female mice, according to transcriptomic microarray analyses, corrected p-value = 0.019. b Relative ccl3 and p16^ink4a mRNA levels in liver of 23-month-old Itpr2 WT (n = 14) and KO (n = 15) female and male mice. Mean ± SEM. Unpaired two-tailed Mann–Whitney U Test. c Micrographs of liver sections and quantification of p16^INK4A-positive cells, stained by immunohistochemistry (IHC) in the livers of 23-month-old mice Itpr2 WT (n = 4) and KO (n = 4) female mice. Mean ± SEM. Unpaired two-tailed Student t-test (*p = 0.0331). d–e Linear regression analyses between intrahepatic fibrosis level or blood AST level and p16^ink4a mRNA levels in the livers of 23-month-old female and male mice (n = 14). Two-tailed Spearman Rank Correlation test. f Linear regression analysis between the number of lipid droplets and p16^ink4a mRNA levels in the liver of 23-month-old female mice (n = 7). Two-tailed Spearman Rank Correlation test. g Quantification of SA-ß-galactosidase-positive cells in Itpr2 WT (n = 8) and KO (n = 7) MEFs at early and late passage. Mean ± SEM. Early vs. Late: Paired two-tailed Student t-test (ns: non-significant). Late WT vs. Late KO: Unpaired two-tailed Welch’s t-test. h p16^ink4a mRNA level in WT (n = 5) and KO (n = 5) MEFs at late passage. Mean ± SEM. Unpaired two-tailed Welch’s t-test. i p16^INK4A, Itpr2 and tubulin protein levels in Itpr2 WT and KO MEFs at late passage. j Inflammatory response gene ontology extracted from microarray of Itpr2 WT (n = 3) and KO (n = 3) MEFs at early and late passage, corrected p-value = 2.292⁻¹². *p < 0.05; **p < 0.01; ***p < 0.001; ns non-significant.

Fig. 3. Loss of Itpr2 diminishes contacts between mitochondria and the ER.

a Proximity ligation assay (PLA) using VDAC1 (outer mitochondrial membrane) and ITPR1 (ER membrane) antibodies in the livers of 23-month-old Itpr2 WT (n = 4) and KO (n = 4) female mice. Dots are formed when the distance between ITPR1 and VDAC1 is below 50 nm. Representative pictures (scale bar: 5 µm) and quantification shown as mean ± SEM. Unpaired two-tailed Student t-test. b Linear regression analysis between the number of MERCs and the percentage of p16^INK4Apositive cells in the liver of 23-month-old Itpr2 WT (n = 4) and KO (n = 4) female mice. Two-tailed Spearman Rank Correlation test. c PLA using Vdac1 and Itpr1 antibodies in early and late passage Itpr2 WT and KO MEFs. Representative pictures (scale bar: 5 µm) and mean ± SEM of n = 10 fields, examined over n = 4 independent experiments. Two-way ANOVA. Tukey’s multiple comparisons test. d Quantification of the number of MERCs normalized to mitochondria number in late passage Itpr2 WT and KO MEFs. MERCs are defined by a distance <50 nm between ER and OMM membranes, evaluated by transmission electron microscopy. Mean ± SEM of n = 20 cells. Unpaired two-tailed Student t-test. e Mean ER-mitochondria distance in MERCs in late passage Itpr2 WT and KO MEFs, based on electron microscopy study. Mean ± SEM of 20 cells, n representing individual MERCs of Itpr2 WT (n = 146) and KO (n = 138) MEFs examined over n = 3 independent biological replicates. Two-tailed Mann–Whitney U Test. f Linear regression analysis between the number of MERCs and percentage of SA-ß-galactosidase positive cells at different passages of Itpr2 WT (n = 4) and KO (n = 4) MEFs. Two-tailed Spearman Rank Correlation test. *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 4. Inducing MERCs promotes premature senescence.

a Graphical representation of the artificial genetic linker to induce MERCs formation. OMM outer mitochondrial membrane; MIS mitochondrial Intermembrane Space. b Using transmission electron microscopy, MERCs number was calculated in Ctrl/Linker-infected MRC5 cells. Mean ± SEM of n = 10 (Ctrl) and n = 11 (Linker) cells. Unpaired two-tailed Student t-test. c Mean ER-mitochondria distance in Ctrl/Linker-infected MRC5 cells. Mean ± SEM of n = 53 (Ctrl) and n = 80 (Linker) MERCs. Unpaired two-tailed Student t-test. d Mitochondrial membrane length associated with ER, depending on the distance (0-50 nm) between mitochondria and ER in Ctrl/Linker-infected MRC5 cells. Mean ± SEM of n = 53 (Ctrl) and n = 80 (Linker) MERCs, representative of n = 3 independent experiments. Multiple t-tests. e Steady-state mitochondrial calcium levels in Ctrl/Linker-infected cells. n: number of analyzed RFP-positive cells representative of n = 3 independent experiments. Mean ± SEM. Two-tailed Mann–Whitney U Test. f Crystal violet staining for Ctrl/Linker-infected MRC5 cells 12 days after infection, representative of n = 3 independent experiments. g Quantification of EdU-positive cells for Ctrl/Linker-infected MRC5 cells. Mean ±  SEM of n = 3 independent experiments. Paired two-tailed Student t-test. h Quantification of SA-ß-galactosidase-positive cells in Ctrl/Linker-infected MRC5 cells. Mean ± SEM of n = 4 independent experiments. Paired two-tailed Student t-test. i RT-qPCR representing relative p16^INK4A, CCL3, IL8, and IL1-ß mRNA levels in Ctrl/Linker-infected MRC5 cells. Mean ± SEM of n = 7 independent experiments. Unpaired two-tailed Welch’s t-test. j Single-cell analysis on RFP-positive Ctrl/Linker-infected cells of mitochondrial membrane depolarisation using JC1 probe. n: number of analyzed cells, representative of n = 3 independent experiments. Box plots represent the first quartile, median, and third quartile, whiskers corresponding to min/max values. Two-tailed Mann–Whitney U Test. k Single-cell analysis of RFP-positive Ctrl/Linker-infected cells of mitochondrial ROS fluorescence. n: number of analyzed cells, representative of n = 3 independent experiments. Box plots represent the first quartile, median, and third quartile, whiskers corresponding to min/max values. Two-tailed Mann–Whitney U Test. l Crystal violet staining for Ctrl/Linker-infected MRC5 cells treated with vehicle (Veh) or NAC, representative of n = 3 independent experiments. m Quantification of SA-ß-galactosidase-positive cells in Ctrl/Linker-infected MRC5 cells, treated with Veh or NAC. Mean ± SEM of n = 3 independent experiments. Two-way ANOVA. Paired Tukey’s multiple comparisons test. n RT-qPCR representing relative p16^INK4A, CCL3, IL8, and IL1-ß mRNA levels in Ctrl/Linker-infected MRC5 cells treated with Veh or NAC. Mean ± SD, representative of n = 3 independent experiments. Two-way ANOVA. Tukey’s multiple comparisons test. * p < 0.05; **p < 0.01; ***p < 0.001.