Acyl-CoA-binding protein as a driver of pathological aging

Abstract

The tissue hormone acyl coenzyme A-binding protein (ACBP, encoded by the gene diazepam-binding inhibitor, DBI) has been implicated in various facets of pathological aging. Here, we show that ACBP plasma concentrations are elevated in (close-to-)centenarians (mean ± SD age 99.5 ± 4.5 y) commensurate with their health deterioration, correlating with a reduced glomerular filtration rate and a surge in senescence-associated cytokines. ACBP neutralization by means of a monoclonal antibody (mAb) improved health span in a strain of progeroid mice. In a mouse model of chronic kidney injury induced by cisplatin, anti-ACBP mAb administration counteracted both histopathological and functional signs of organ failure. ACBP inhibition also prevented the senescence of tubular epithelial cells and glomerular podocytes induced by cisplatin or doxorubicin, respectively, as measurable by the immunohistochemical detection of cyclin-dependent kinase inhibitor 1A (CDKN1A, best known as p21). Senescence was also prevented by anti-ACBP mAb treatment in additional mouse models of accelerated aging. This applied to liver damage induced by a combination of high-fat diet and carbon tetrachloride, where hepatic cells become senescent. Moreover, administration of anti-ACBP mAb prevented natural and doxorubicin-accelerated cardiomyocyte senescence. We performed single-nucleus RNA sequencing to study the transcriptome of hearts that had been exposed to doxorubicin and/or anti-ACBP in vivo. In cardiomyocytes, doxorubicin caused an anti-ACBP-reversible dysregulation of mRNAs coding for cardioprotective proteins involved in autophagy, fatty acid oxidation, mitochondrial homeostasis, and oxidative phosphorylation. Altogether, these findings plead in favor of a broad age-promoting effect of ACBP across different organ systems.

Keywords: aging; autophagy; chemotherapy; kidney injury; senescence.

Fig. 1.

ACBP is elevated in (close-to-)centenarians and increases further with disease.(A) A cohort of N = 37 participants over 90 y old was recruited at their nursing home during a “normal day”, i.e., in a context of relative functional autonomy (N = 16), or during episodes of acute hospitalization at the Gregorio Marañón General University Hospital (N = 21). They were matched in silico to healthy young volunteers from the DESIR cohort. (B) Plasma ACBP levels measured in the (close-to-)centenarians are higher, as compared to young controls, and are highest in those who belong to the hospital group. (C) Plasma ACBP levels are positively correlated with the CCI. (D) Plasma ACBP levels are negatively correlated with glomerular filtration rate (GFR), an indicator of renal clearance. (E) ACBP, along with a pool of 12 proinflammatory cytokines (out of the 40 plasma cytokines measured by proximity extension assay), is higher in the patients from the hospital group. (F) Hierarchically clustered correlation matrix highlights a cluster of inflammatory cytokines positively correlating with ACBP within all patients.(G) Correlation analysis of the hospital and nursing home groups separately shows that three plasma cytokines consistently positively correlate with ACBP: vascular endothelial growth factor A (VEGFA), oxidized low-density lipoprotein receptor 1 (OLR1) and hepatocyte growth factor (HGF). Comparisons between groups were performed by one-way ANOVA for plasma levels of ACBP between the three groups and by the Wilcoxon signed-rank test followed by FDR correction for multiple comparisons for comparisons of log2-normalized values of plasma cytokines. Pearson’s product–moment correlation coefficients and the corresponding asymptotic P-values were used for the heatmap and Venn diagram. ACBP: acyl-CoA-binding protein; CCL(2–19): C-C motif chemokines (2–19); CSF1: macrophage colony-stimulating factor; CSF2: granulocyte–macrophage colony-stimulating factor; CSF3: granulocyte colony-stimulating factor; CXCL(8–12): C-X-C motif chemokine ligand (8–12); EGF: epidermal growth factor; FLT3LG: Fms-related tyrosine kinase 3 ligand; IFNG: interferon gamma; IL(2–27): interleukin (2–17); LTA: lymphotoxin-alpha; MMP(1/12): matrix metalloproteinase-(1/12); OSM: oncostatin M: TGFA: transforming growth factor alpha; TNF: tumor necrosis factor; TNFSF10: TNF-related apoptosis-inducing ligand; TNFSF12: TNF ligand superfamily member 12.

Fig. 2.

ACBP neutralization improves health span in the Zmpste24 ^−/− murine progeria model. (A) In-house bred C57Bl/6 N mice deficient for the Zmpste24 metalloprotease gene were randomized at 8 wk of age to be treated either with anti-ACBP (n = 15 females, n = 9 males) or mouse IgG2a isotype control (n = 13 females, n = 9 males) monoclonal antibodies (2.5 mg/kg i.p.), both administered i.p. twice weekly. Functional evaluation was performed after 21 wk of treatment. (B) Representative pictures of the general state of the animals at 26 wk of age. (C) Representative images of computed tomography scanner (CT-scan) from mice of the different groups, and associated measurements of the spine curvatures. (D) Representative images of spleen morphology and associated measurement of the spleen weights in the different groups, normalized by total body mass. (E) Blood measurements of hematocrit (Hct) and calculated hemoglobin concentration (cHgb). (F) Scores of severity for kyphosis, teeth damage, hair loss, loss of walking ability were assessed blindly. The results presented are pooled from two independent mouse cohorts. (G) Spasm prevalence within each group, as assessed by a 5-s tail suspension test. The effect of anti-ACBP on each of the functional criteria was assessed either separately for males and females or in the total cohort of mice by the Wilcoxon rank-sum test or t test depending on variable normality.

Fig. 3.

Renal damage induced by chronic administration of cisplatin is dampened by ACBP-neutralizing monoclonal antibodies. (A) Chronic kidney disease was induced in adult male C57Bl/6 J mice by 4 wk administrations of cisplatin (8 mg/kg i.p., compared to vehicle-injected controls, CTR). In half the animals of the cohort, a monoclonal antibody against ACBP was administered on the day prior to the first cisplatin injection (Anti-ACBP, 5 mg/kg i.p., compared to isotype-injected controls) and maintained weekly for 8 wk. (B and C) At the end of the experiment, blood urea nitrogen (BUN) was measured, and the estimated glomerular filtration rate (eGFR) was calculated from the blood parameters. (D and E) Representative images of hematoxylin–eosin (HE) and periodic acid–Schiff (PAS) staining of kidney sections show the structural changes induced by cisplatin. (F) Pathological scoring of the renal damage was performed on a scale from 0 to 4 based on tubular necrosis, brush border loss, tubular dilation, tubular cast formation, widening of the interstitium, degeneration, and inflammation by two independent examiners, and the average score was calculated for each mouse. (G and H) Masson’s trichrome staining was used to quantify renal fibrosis by automatic segmentation of the blue color. Statistical comparisons were done by linear mixed modeling of each measurement (eGFR, pathological score, % fibrotic area), testing the interaction of two fixed effects (cisplatin and anti-ACBP) and taking mouse identity and technical parameters (examinator, histological section, field) as random effects. P-values were computed for the chosen pairwise contrasts with the estimated marginal means method.

Fig. 4.

The organ-protective functions of ACBP neutralization are accompanied by a reduction of the senescence marker p21 in various preclinical models. (A) Representative images of the immunohistochemical (IHC) detection of p21 (CDKN1A: cyclin-dependent kinase inhibitor 1A) positive nuclei in kidney sections from cisplatin-treated mice, and (B) the corresponding quantifications show that the cisplatin-induced increase in p21 nuclear staining is reduced by anti-ACBP administration. (C) Representative images of the immunoblotting of protein lysates from full kidneys. (D) Quantification of the p21 signal after normalization by the stably expressed glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loading control, relative to control group expression levels. (E) Experimental scheme of a model of anthracycline-induced accelerated aging, in which mice were injected weekly with doxorubicin (5 mg/kg, i.p., compared to PBS vehicle-injected controls) with or without anti-ACBP monoclonal antibody (5 mg/kg, i.p., compared to isotype-injected controls) for four cycles. Similar to the results obtained with cisplatin, p21 signal augments in the kidney glomeruli (F and G) and heart cardiomyocytes (H and I) upon doxorubicin treatment and is completely abrogated by anti-ACBP. (J) Experimental scheme of chronic liver damage induced by prolonged administration of western diet (WD) and carbon tetrachloride (CCl₄), compared to regular chow diet (RCD) and vehicle (olive oil) injections. (K and L) Nuclear staining of p21 in hepatocytes by IHC is increased in this model of liver damage, and reversed by anti-ACBP treatment. The interaction between the effect of organ-damaging treatments (cisplatin, doxorubicin, or WD + CCl4) and anti-ACBP treatment was evaluated by two-way ANOVA, followed by estimation of the marginal means for pairwise comparisons.

Fig. 5.

Single-nuclei RNA sequencing analysis of the reversion of the doxorubicin-induced cardiac damage upon ACBP neutralization. Nuclei were extracted from ventricles from mice treated with chronic administration of doxorubicin with or without anti-ACBP (scheme in Fig. 4E) and submitted to single-nuclei RNA-sequencing (snRNA-seq, N = 2 per experimental condition). (A) 2D projection of the first two dimensions from the Uniform Manifold Approximation Projection (UMAP) of the 14 clusters, corresponding to cell populations, were identified based on their nuclear transcription profiles. (B) In the top 8 abundant clusters, the amplitude of transcriptional changes induced by DOXO was quantified as the number of differentially expressed genes (DEGs, Wilcoxon test P < 0.05 and fold-change > 1.5) normalized by the number of cells in the clusters. (C) Volcano plots show the transcriptional changes induced in the cardiomyocytes cluster by DOXO alone or by the combination of anti-ACBP with DOXO, both compared to control conditions. (D) Venn diagram comparison of the corresponding DEGs was used to define three categories of DEGs: genes that are differentially expressed (DE) by DOXO but not anymore when combining it to anti-ACBP (“reversible DEGs”), genes that are specifically DE in the anti-ACBP + DOXO combination (“specific DEGs”), and genes that are DE by DOXO no matter the monoclonal antibody treatment (“common DEGs”). (E and F) Pathway enrichment analysis of the reversible DEGs and specific DEGs show enrichment of metabolic and cardioprotective pathways from the Gene Ontology (Bioprocesses terms, abbreviated G) and Kyoto Encyclopedia of Genes and Genomes (KEGG, abbreviated K) databases. Numbers to the right of each pathway name correspond to the number of upregulated (green) and downregulated (red) genes from the pathway found in the DEGs list.