A high-fat diet and NAD(+) activate Sirt1 to rescue premature aging in cockayne syndrome

Abstract

Cockayne syndrome (CS) is an accelerated aging disorder characterized by progressive neurodegeneration caused by mutations in genes encoding the DNA repair proteins CS group A or B (CSA or CSB). Since dietary interventions can alter neurodegenerative processes, Csb(m/m) mice were given a high-fat, caloric-restricted, or resveratrol-supplemented diet. High-fat feeding rescued the metabolic, transcriptomic, and behavioral phenotypes of Csb(m/m) mice. Furthermore, premature aging in CS mice, nematodes, and human cells results from aberrant PARP activation due to deficient DNA repair leading to decreased SIRT1 activity and mitochondrial dysfunction. Notably, β-hydroxybutyrate levels are increased by the high-fat diet, and β-hydroxybutyrate, PARP inhibition, or NAD(+) supplementation can activate SIRT1 and rescue CS-associated phenotypes. Mechanistically, CSB can displace activated PARP1 from damaged DNA to limit its activity. This study connects two emerging longevity metabolites, β-hydroxybutyrate and NAD(+), through the deacetylase SIRT1 and suggests possible interventions for CS.

Fig. 1. A high fat diet rescues the metabolic phenotype of Csb^m/m mice

(A) Body weights of WT and Csb^m/m mice on various diets; SD: Standard diet; HFD: High fat diet; Resv: resveratrol supplemented standard diet; CR: Caloric restricted (n=12–14, mean ± SEM). (B) Feeding efficiency shown by weight gain per food intake (n=12–14, mean ± SEM). (C) Whole body oxygen consumption over 72 hours (n=12–14, mean ± SEM). (D) Respiratory exchange rates (n=12–14, mean ± SEM). (E) Heat production (n=12–14, mean ± SEM). (F) Oral glucose tolerance tests performed after 3 hour fasting (n=5–9, mean ± SEM). (G) Insulin levels after 3 hours fasting (n=7–11, mean ± SEM). (H) Glucose levels after 3 hours fast (n=5–9, mean ± SEM). (I) Representative images of liver histology stained with hematoxylin and eosin. (J) Representative images of scanning electron microscopy of the sinusoidal endothelium (triangle: fenestration example, highlighted area: sieve plate). (K) Quantification of the fenestration diameter of the liver sinusoid (n=4–6000 from 3 mice in each group, mean ± SD). (L) The porosity of the liver using the diameter measured in (K) and the total surface area of the endothelium (n=3, mean ± SEM). See figure S1 for further information.

Fig. 2. A high fat diet increases β-hydroxybutyrate levels and rescues the neurological phenotype of Csb^m/m mice

(A) A principal component analysis (PCA) of the unselected average gene expression Z-score from the cerebellum and a hierarchical clustering of the same data (n=3–7). (B) Levels of circulating β-hydroxybutyrate (β-OHB) in the mice (n=6–12, mean ± SEM). (C) Levels of β-OHB in the brain of mice (n=4–9, mean ± SEM). (D) Orthogonal partial least square regression of metabolomics data done on the same samples as in (D) and a list of some altered metabolites when comparing genotype only. (n=3–7). (E) Hearing tests done by exposing the mice to a 108 db recorded clap, videotaping the reaction and subsequently blindly scoring the mice for a reaction to the sound (n=10–14). (F) Representative histological images of hematoxylin and eosin stained sections of the inner ear (blue highlight: spiral ganglion). (G) Quantification of cells in the spiral ganglion (n=3, mean ± SEM). (H) Aerobic capacity of the mice measured by forced treadmill exercise (n=7–11, mean ± SEM). (I) Serum lactate levels (n=7–12, mean ± SEM). (J) Extracellular acidification rates of immortalized CSB patient cells (CS1AN) reconstituted with WT CSB (WT) or an empty vector (CSB) (n=28 separate experiments, mean ± SEM). (K) NAD⁺/NADH ratio (n=3, mean ± SEM).

Fig. 3. PARP1 activation drives SIRT1 depression and the mitochondrial phenotype in CSB deficient cells

(A) Representative confocal microscopy images and quantification of WT and CSB deficient cells stained for PARP1 and PAR (n=3, mean ± SEM). (B) Representative immunoblot of PAR, PARP1, PARG1 and UCP2 in WT and CSB deficient cells (n=3, mean ± SEM). (C) Mass spectrometry of nicotinamide adenine dinucleotide (NAD) and NAD-metabolites in WT and CSB deficient cells; NMN: Nicotinamide mononucleotide; NAM: Nicotinamide; (n=6–8, mean ± SEM). (D) Representative immunoblot of sirtuin levels in WT and CSB deficient cells. (E) Immunoblot of protein levels from the cerebellum of WT and Csb^m/m mice on various diets. Each lane is a separate mouse. (F) FCCP uncoupled respiration normalized to basal respiration under various 24 hour treatments (n=3–28 separate seahorse experiments, mean ± SEM). (G) FCCP uncoupled respiration normalized to basal respiration 72 hours after SIRT1 siRNA treatment (n=3, mean ± SEM). (H) FCCP uncoupled respiration normalized to basal respiration 72 hours after UCP2 overexpression (n=3, mean ± SEM). (I) Flow cytometry of WT and CSB deficient cells after overexpression of UCP2 and stained with tetramethylrhodamine methyl ester (TMRM) for membrane potential and mitosox for mitochondrial superoxide production (n=3, mean ± SEM).

Fig. 4. β-hydroxybutyrate and PARP inhibition rescues the CS phenotype through SIRT1 activation

(A) FCCP uncoupled respiration normalized to basal respiration 48 hours after 10 mM β-OHB treatment (n=3 separate seahorse experiments, mean ± SEM). (B) Representative immublot of treatment of WT and CSB deficient cells with increasing concentrations of β-OHB. (C) Flow cytometry of WT and CSB deficient cells treated with β-OHB and or EX-527 for 48 hours and stained with mitosox (n=6, mean ± SEM). (D) Oxygen consumption rate (OCR) relative to extracellular acidification rate (ECAR) (n=3–12 separate seahorse experiments, mean ± SEM). (E) Acetyl-CoA levels after treatment with the PARP inhibitor PJ34 or β-OHB for 24 hours (n=6, mean ± SEM). (F) Representative immunoblot of acid extracted histones after treatment with the PARP inhibitor PJ34 or β-OHB for 24 hours. (G) Representative immunoblot from WT and CSB deficient cells after 24 hours treatment with MB-3, NU9056 or increasing concentration of L002 and flow cytometry of WT and CSB deficient cells treated with MB-3, NU9056 or increasing concentration of L002 for 24 hours and stained with mitosox (n=3–12, mean ± SEM). (H) Representative immunoblot of protein levels after knockdown of various proteins. (I) Flow cytometry of WT and CSB deficient cells subjected to siRNA knockdown of various proteins and stained with mitosox (n=3, mean ± SEM). (J) Representative immunoblot of protein levels after treatment with L002 and/or β-OHB. (K) Representative immunoblot of protein levels after treatment with L002 and/or MG-132.

Fig. 5. The longevity effect of β-hydroxybutyrate and PARP inhibition are non-additive in short-lived csb-1 nematodes

(A) Representative immunoblot from old and young csb-1 and N2 worms. Each lane represents a separate worm cohort. (B) Kaplan-Meier survival curves of csb-1 and N2 nematodes (n=100). (C) Survival curves of csb-1 nematodes treated with 0.1 µM PJ34 (n=50). (D) Survival curves of csb-1 nematodes treated with 25 mM β-hydroxybutyrate (β-OHB, n=50). (E) Survival curves of csb-1 nematodes treated with 0.1 µM PJ34 and 25 mM β-OHB ( n=50). (F) Survival curves of csb-1 nematodes treated with 0.1 µM PJ34, 25 mM β-OHB or 0.1 µM PJ34 and 25 mM β-OHB (n=50). (G) Survival curves of WT N2 nematodes treated with 0.1 µM PJ34 (n=50). (H) Survival curves of WT N2 nematodes treated with 25 mM β-OHB (n=50). (I) Survival curves of WT N2 nematodes treated with 0.1 µM PJ34 and 25 mM β-OHB (n=50). (J) Survival curves of WT N2 nematodes treated with 0.1 µM PJ34, 25 mM β-OHB or 0.1 µM PJ34 and 25 mM β-OHB (n=50). (K) Survival curves of WT N2 and csb-1 nematodes treated 0.1 µM PJ34 and 25 mM β-OHB (n=50).

Fig. 6. CSB inhibits PARP1 activation in vitro and in vivo through displacement of PARylated PARP1 from DNA

(A) Electromobility shift assay (EMSA) of PARP1 and CSB binding to double stranded DNA. (B) BamH1 incision of a 42-mer oligo preincubated with 250 nM PARP1 and with increasing amounts of CSB with or without NAD⁺ (n=3, mean ± SEM). (C) Representative immunoblot and quantification of in vitro poly-ADP-ribosylation (PARylation) of CSB and PARP1. Reactions were performed with recombinant proteins in the presence of undamaged or damaged DNA (n=3, mean ± SEM). (D) Representative immunoblot of whole cell (PARylation) after 5 J/m² treatment in WT and CSB deficient cells at various timepoints. (E) Recruitment of gfp-tagged CSB to laser induced DNA damage after 1 hour preincubation with PARP inhibitors 3AB or PJ34. (F) Representative slot-blot showing non-covalent interaction between CSB and PAR. (G) The PAR binding motif in CSB. (H) Recruitment to laser induced DNA damage of gfp-tagged WT CSB or CSB harboring K to A mutations in the 4 conserved lysines. (I) Recruitment of gfp-tagged WT CSB to laser induced DNA damage in WT HeLa or PARP1^−/− HeLa cells.

Fig. 7. PARP inhibition or NAD⁺ replenishment rescues CS associated alterations in mice and human cells

(A) Measurements of the oxygen consumption rate using the Seahorse XF24 analyzer while adding increasing doses of the PARP inhibitors 3AB, NU1025 or PJ34 (n=3 separate experiments, mean ± SEM). (B) Flow cytometry of mitochondrial superoxide production in WT and CSB deficient cells after 24 hours treatment with the PARP inhibitors (n=3, mean ± SEM). (C) Whole body oxygen consumption rates in 4 months (young) and 16 months (old) old WT and Csb^m/m mice after daily intraperitoneal injections of PJ34 (25mg/kg body weight) (n=3–8, right is the quantification of the slopes). (D) and (E) NAD⁺ and ATP levels in the cerebellum of young and old WT and Csb^m/m mice after 1 week treatment with the NAD⁺ precursor nicotinamide riboside (n=3–5, mean ± SEM). (F–I) Mebrane potential and ROS production in isolated mitochondria from the cerebellum of WT and Csb^m/m mice (n=3–5, mean ± SEM). (J) A principal component analysis and hierarchical clustering of transcriptomic changes in the cerebellum of mice treated with NR. (K) Venn-diagram of significantly changed gene expressions when comparing old Csb^m/m saline treated mice vs. old WT saline treated mice and old Csb^m/m NR treated mice vs. old WT saline. (L) Venn-diagram of significantly changed gene expressions when comparing old Csb^m/m saline treated mice vs. old WT saline treated mice and old Csb^m/m NR treated mice vs. old WT saline.