NAD+ supplementation prevents STING‐induced senescence in ataxia telangiectasia by improving mitophagy

Abstract Senescence phenotypes and mitochondrial dysfunction are implicated in aging and in premature aging diseases, including ataxia telangiectasia (A‐T). Loss of mitochondrial function can drive age‐related decline in the brain, but little is known about whether improving mitochondrial homeostasis alleviates senescence phenotypes. We demonstrate here that mitochondrial dysfunction and cellular senescence with a senescence‐associated secretory phenotype (SASP) occur in A‐T patient fibroblasts, and in ATM‐deficient cells and mice. Senescence is mediated by stimulator of interferon genes (STING) and involves ectopic cytoplasmic DNA. We further show that boosting intracellular NAD+ levels with nicotinamide riboside (NR) prevents senescence and SASP by promoting mitophagy in a PINK1‐dependent manner. NR treatment also prevents neurodegeneration, suppresses senescence and neuroinflammation, and improves motor function in Atm−/− mice. Our findings suggest a central role for mitochondrial dysfunction‐induced senescence in A‐T pathogenesis, and that enhancing mitophagy as a potential therapeutic intervention.

GGGCGCCCGGTTCTT, and R: CCTCGTCCTGCAGTTCATTCA. The Atm -/mice and their age-and sex-matched littermates were used for behavioral studies. All animal experiments were performed and approved by NIA Animal Care and Use Committee.

Behaviors
For behavioral tests, 1.5-month-old mice were given NR at 3.5 mg/ml (12 mM) in their drinking water while the control groups were received only drinking water for 2 months.
In the open field test, each individual mouse was placed in the center of open field box (40 cm×40 cm×40 cm) (Med Associates, Georgia, VT, USA) and recorded for 20 minutes. The tracking software (Activity monitor version 4, Med-Associates) recorded the exploratory behavior. The apparatus was cleaned with 70% ethanol before testing the next mouse. Travel distance, number and duration of jump and rearing parameters were recorded and analyzed. In Digi-gait test, mice were habituated for 5 minutes.
Animals were recorded by collecting digital images at 80 frames per second with a highspeed video camera located beneath a transparent treadmill moving at a speed of 15 cm/s (Digi-Gait Imaging Systems, Boston, MA). A variety of gait parameters were analyzed R software.

NAD + detection
NAD + and NADH were measured with a commercially available NAD + /NADH assay kit (Abcam,#ab65348) according to the manufacturer's protocol. For cells, 4 X 10 5 cells homogenized in 400 µl lysis buffer. For mice tissue, 20 mg were homogenized in 500 µl lysis buffer and clarified at 17,000 g for 5 min at 4 °C. Supernatants were filtered using 10 kD filters (Millipore) and spun at 17,000 g for 60 min. To measure NADH, NAD + was decomposed by incubation at 65 °C for 30 min. Standard curves (5-200 pg/ml) were generated for quantification.

ATP detection
Twenty milligrams of mice cerebellar samples or 4 X 10 5 cells were harvested, and ATP levels were measured with a commercially available ATP Assay Kit (Colorimetric/Fluorometric) (Abcam, ab83355) according to the manufacturer's protocol.

RNA purification
Twenty milligrams of mice cerebellar cortex or 4 X 10 5 cell pellets were homogenized and RNA was extracted purified with PureLink™ RNA isolation kit following manufacturer's protocol (Thermo). RNA concentration and purity were conducted using a NanoDrop ND-1000 spectrophotometer.

Microarray
The quality of the RNA was inspected using a 2100 Bioanalyzer (Agilent Technologies).
Samples with RNA integrity less than 7.5 were discarded. The microarray was performed by the Gene expression and Genomics core facility (NIA) and analyzed using DIANE 6.0 software as described before. A gene was considered significant if the absolute value of its absolute value of z-ratio ≥1.5, p-value ≤ 0.05, false discovery rate ≤ 0.30, as well as sample group independent ANOVA test p ≤ 0.05. A complete set of 880 canonic pathways and 2392 chemical perturbation gene sets were obtained from the Molecular Signatures Database (MSigDB, Broad Institute, Massachusetts Institute of Technology, MA, USA). The complete set was tested for Geneset enrichment using parametric analysis of gene set enrichment (PAGE). Raw microarray hybridization intensity data were filtered to remove undetected and array control probes, before computing log z-score for each sample to identify and exclude possible outlying samples via clustering, scatter plots, and principal components analysis. In A-T fibroblasts, we excluded HT5 and AT5 based on above criteria. After removal of the outlying samples, a sample-specific quantile normalization of the filtered raw average signal was employed to yield quantile normalized log scale z-scores. These quantile zscores were used to ensure a standard normal distribution in further statistical analysis, including ANOVA, t-and z testing. |Z-ratio| ≥1.5 were used as cut-off values and calculated using a combination of thresholds, including z-test p < value 0.05, FDR ≤ 0.30, and average signal intensity of comparison>0, as well as global sample ANOVA Ftest p ≤ 0.05. The gene expression change z-ratio values were then used as input to perform PAGE testing. For each gene set change, an aggregate z-score, and p-value with false discovery change was calculated and reported for statistical significance. The accession number for the raw and processed microarray data reported in this paper is GSE152289.

Quantitative real-time PCR
cDNA was synthesized using iScript cDNA Synthesis kit (BioRad) and qPCR analysis was done with power SYBR Green PCR master mix (Thermo Fisher). The primers used to amplify each transcript are as Table S2.

Immunofluorescence
Anesthetized mice were first perfused with cold PBS and then with 4% paraformaldehyde (PFA) in PBS. The collected brains were then placed in 30% sucrose in PBS (Sigma) until sunken at 4 °C. 1:8 series equidistant floating 30-μm coronal sections (interval 240 μm) were obtained with a cryostat CM 3080S (Leica).
Approximately 9-10 slices of each mouse were incubated in blocking buffer (5% goat serum and 0.3% Triton X-100 in PBS) for 60 min at room temperature (RT). Thereafter, brain slices were incubated overnight with the primary antibody at 4°C and then incubated with the appropriate fluorescent probe conjugated secondary antibodies for 1 h at RT protected from light in a humid box. Nuclei were stained with DAPI at 1:5000 dilution for 20 min. The pictures were taken using a Zeiss 880 LSM confocal microscope (Zeiss).

Respirometry on cells or primary neurons
4 X 10 4 cells or neurons per well were seeded on Seahorse culture plate. Basal and uncoupled oxygen consumption rates (OCRs) and the extracellular acidification rate (ECAR) were measured using the Seahorse extracellular flux bioanalyzer (XF96, Seahorse Bioscience Inc.. To uncouple mitochondria, 2 μM of FCCP was injected after a basal respiration measurement. All measurements were performed in 4 or 5 replications and results were normalized to protein level.

Western blotting
Western blotting was performed according to manufacturer's instructions. Briefly, cell pellets or tissue were lysed using 1x RIPA buffer (Sigma-Aldrich, R0278-50ML) containing protease inhibitor cocktail (Sigma-Aldrich, P8340-5ML) and phosphatase inhibitor cocktail 1 (Sigma-Aldrich, P2850-5ML). Protein concentration were measured with pierce™ BCA protein assay kit (Thermo fisher, 23225). 20 µg samples were loaded and separated on 4-12% Bis-Tris gel (Thermo Fisher Scientific, #NP0336BOX) and transferred to PVDF membranes. Membranes were blocked in TBST + 5% milk, incubated overnight with primary antibody, washed in TBST, incubated with HRPconjugated secondary antibody for 60 min. Immunoreactivity was detected with an ECL kit (Millipore, or Thermo). Optical density of the immunoreactivity bands was analyzed using Image J software (NIH). All secondary antibodies were obtained from Cell signaling.

SA-β-galactosidase assay
Senescence-associated β-galactosidase (SA-β-gal) activity was detected using the senescence β-galactosidase staining kit (Cell signaling), according to manufacturer's instructions. For cerebellar slices, staining for SA-β-Gal was performed on free-floating mouse brain sections in the SA-β-gal staining solution at 37°C for 16-20 h. For cells, cells were fixed with 4% formaldehyde in PBS for 15min at room temperature. The fixed cells were incubated with SA-β-Gal staining solution at 37 °C for 16 h after PBS washing. The percentages of positively stained cells were calculated based on three replicates.

Rat neurons culture and treatments
Cerebral cortex tissue was collected from Sprague-Dawley rat embryos at 18 days of gestation. Dissociated cells were seeded into polyethyleneimine-coated plastic dishes or slide chambers in Neurobasal Plus Medium (Thermo fisher, A3582901) supplemented with 2% B27 Plus Supplement (Thermo fisher Scientific, A3582801) and 1% Anti-Anti (Gibco) at a density of 80,000 cells/cm 2 . The neurons were grown in 20% O2 + 5% CO2 at 37°C for further experiments. Rat cortical neurons at 0 day in vitro (DIV) were treated with either DMSO or 1µm ATM inhibitor KU-60019 (Sigma, SML1416-25MG) then treated for 72 h, then change with fresh medium with treatment on 3 DIV and 6 DIV. On 6 DIV, neurons were treated with dH2O or Nicotinamide riboside/NR, 1mM. Then on 7 DIV the assays for the multiple endpoints were performed.

Mitochondrial parameters assay
For A-T cells and human SH-SY5Y were incubated with different dyes (all from Life Technologies TM ), including TMRM (40 nM for 15 min) to detect mitochondrial membrane potential, MitoTracker Green (50 nm for 15 min) for mitochondrial content, and mitoSOX (3 μM for 30 min) for mitochondrial ROS, followed by detection by flow cytometer (BD LSRFortessa™). Median number was recorded and analyzed by FlowJo™ v10.6.2.

Subcellular fractionation
Subcellular fractionation and mitochondrial DNA quantification was performed as follows: 5x10 6 HT1/AT1 or 8x10 6 SH-SY5Y cells were lysed in 1ml Mitochondrial isolation buffer (225 mM Mannitol, 75 mM Sucrose, 5 mM HEPES, 1 mg/ml Fatty acid free-BSA, Protease and phosphatase inhibitors, adjust pH 7.4). Samples were centrifuged at 2,200xg for 3 min at 4°C. Pellet are nuclear fractions. Supernatants were transferred to fresh tubes and centrifuged at 17,000xg for 10min at 4C, transferring supernatants to fresh tubes between centrifugation steps to finally yield cytoplasmic fractions. 10 ng cytoplasmic DNA was used for qPCR analysis of mitochondrial DNA using gene-specific primers; nuclear gene RPL13A was quantified from the respective 10ng from nuclear fraction for normalization. Primers are shown as Table S2.

Statistical analysis
The sample size (n) for each experimental group is described in each figure legend.
Either Statview 5.0 or GraphPad Prism software was used for statistical analyses.
Quantitative data displayed as histograms are expressed as means ± standard error of the mean or means ± standard deviation (represented as error bars). Results from each group were averaged and used to calculate descriptive statistics. Student's t test was used for comparisons between two groups. Statistical significance was set at a P value of *p < 0.05, **p < 0.01 and ***p < 0.001.

Table S1
Genes with the largest enrichment score in M. musculus.

Table S2
List of primers used in M. musculus and human cells.   Fig. S2C (D). (E) Mitochondrial ROS, mitochondrial content, mitochondrial membrane potential, and cellular ROS were measured by flow cytometry in SH-SY5Y shCtrl and shATM cells. n = 8-9 cell cultures in each group. Data are shown as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 by two-tailed Student's t test.