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

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.

Inhibitory effect of alpha-lipoic acid on mitochondrial dysfunction and interleukin-8 expression in interleukin-1beta-stimulated ataxia teleangiectasia fibroblasts.

Ataxia telangiectasia (A-T) is an inherited neurodegenerative disease caused by mutation in the ataxia telangiectasia mutated (ATM) gene, leading to loss of function in the encoded protein ATM. Because ATM functions to reduce oxidative stress by up-regulating antioxidant enzymes, oxidative stress is a prevalent A-T phenotype and a mediator of the inflammation that drives A-T pathology. Reactive oxygen species (ROS) levels and the expression of pro-inflammatory cytokine interleukin-8 (IL-8) were higher in A-T cells than in normal cells. ROS are related to mitochondrial dysfunction and activation of nuclear factor kappa B (NF-κB) to induce IL-8 expression. Alpha-lipoic acid (α-LA), a naturally occurring thiol compound, shows an antioxidant effect in various cells. This study is aimed to determine if α-LA confers protection against NF-κB activation, IL-8 expression, and mitochondrial dysfunction in A-T cells which are exposed to the inflammatory cytokine IL-1ß. A-T fibroblasts were treated with or without α-LA. The levels of intracellular and mitochondrial ROS, mRNA and protein levels of IL-8, mitochondrial membrane potential (MMP), ATP levels, and DNA binding activity of NF-κB were determined. As a result, IL-1ß increased NF-κB activation, IL-8 expression, intracellular and mitochondrial ROS levels, but decreased MMP and ATP level in A-T cells. Pretreatment of A-T cells with α-LA inhibited IL-1ß-induced activation of NF-κB, IL-8 expression, and mitochondrial dysfunction by reducing ROS levels. In conclusion, supplementation with α-LA may be beneficial for reducing the oxidative stress-induced mitochondrial dysfunction and IL-8 production associated with A-T.

Comparison of Selected Parameters of Redox Homeostasis in Patients with Ataxia-Telangiectasia and Nijmegen Breakage Syndrome.

This study compared the antioxidant status and major lipophilic antioxidants in patients with ataxia-telangiectasia (AT) and Nijmegen breakage syndrome (NBS). Total antioxidant status (TAS), total oxidant status (TOS), oxidative stress index (OSI), and concentrations of coenzyme Q10 (CoQ10) and vitamins A and E were estimated in the plasma of 22 patients with AT, 12 children with NBS, and the healthy controls. In AT patients, TAS (median 261.7 µmol/L) was statistically lower but TOS (496.8 µmol/L) was significantly elevated in comparison with the healthy group (312.7 µmol/L and 311.2 µmol/L, resp.). Tocopherol (0.8 µg/mL) and CoQ10 (0.1 µg/mL) were reduced in AT patients versus control (1.4 µg/mL and 0.3 µg/mL, resp.). NBS patients also displayed statistically lower TAS levels (290.3 µmol/L), while TOS (404.8 µmol/L) was comparable to the controls. We found that in NBS patients retinol concentration (0.1 µg/mL) was highly elevated and CoQ10 (0.1 µg/mL) was significantly lower in comparison with those in the healthy group. Our study confirms disturbances in redox homeostasis in AT and NBS patients and indicates a need for diagnosing oxidative stress in those cases as a potential disease biomarker. Decreased CoQ10 concentration found in NBS and AT indicates a need for possible supplementation.

Role for p53 in selenium-induced senescence.

The tumor suppressor p53 and the ataxia-telangiectasia mutated (ATM) kinase play important roles in the senescence response to oncogene activation and DNA damage. It was previously shown that selenium-containing compounds can activate an ATM-dependent senescence response in MRC-5 normal fibroblasts. Here, the shRNA knockdown approach and other DNA damage assays are employed to test the hypothesis that p53 plays a role in selenium-induced senescence. In MRC-5 cells treated with methylseleninic acid (MSeA, 0-10 µM), depletion of p53 hampers senescence-associated expression of ß-galactosidase, disrupts the otherwise S and G2/M cell cycle arrest, desensitizes such cells to MSeA treatment, and increases genome instability. Pretreatment with KU55933, an ATM kinase inhibitor, or NU7026, an inhibitor of DNA-dependent protein kinase, desensitizes MSeA cytotoxicity in scrambled but not p53 shRNA MRC-5 cells. These results suggest that p53 is critical for senescence induction in the response of MRC-5 noncancerous cells to selenium compounds.

Synthesis and evaluation of compounds that induce readthrough of premature termination codons.

A structure-activity relationship (SAR) study was carried out to identify novel, small molecular weight compounds which induce readthrough of premature termination codons. In particular, analogs of RTC13, 1, were evaluated. In addition, hypothesizing that these compounds exhibit their activity by binding to the ribosome, we prepared the hybrid analogs 13 containing pyrimidine bases and these also showed good readthrough activity.

Physical and biomedical countermeasures for space radiation risk.

Radiation exposure represents a serious hindrance for long-term interplanetary missions because of the high uncertainty on risk coefficients, and to the lack of simple countermeasures. Even if uncertainties in risk assessment will be reduced in the next few years, there is little doubt that appropriate countermeasures have to be taken to reduce the exposure or the biological damage produced by cosmic radiation. In addition, it is necessary to provide effective countermeasures against solar particle events, which can produce acute effects, even life threatening, for inadequately protected crews. Strategies that may prove to be effective in reducing exposure, or the effects of the irradiation, include shielding, administration of drugs or dietary supplements to reduce the radiation effects, crew selection based on a screening of individual radiation sensitivity. It is foreseeable that research in passive and active radiation shielding, radioprotective chemicals, and individual susceptibility will boost in the next years to provide efficient countermeasures to the space radiation threat.

Detection of individual differences in radiation-induced apoptosis of peripheral blood lymphocytes in normal individuals, ataxia telangiectasia homozygotes and heterozygotes, and breast cancer patients after radiotherapy.

Quantification of radiation-induced apoptosis in peripheral blood lymphocytes (PBLs) has been proposed as a possible screening test for cancer-prone individuals and also for the prediction of normal tissue responses after radiotherapy. We have used the TUNEL assay (terminal transferase nick-end labeling) 24 h after irradiation with 4 Gy at high dose rate to assess interindividual differences in radiation-induced apoptosis between (1) a panel of normal individuals, (2) ataxia telangiectasia (AT) homozygotes and heterozygotes, and (3) breast cancer patients who had received radiotherapy 8-13 years ago, including a number of patients who had suffered adverse responses to radiation. With this protocol, we show clear differences in radiation-induced apoptosis between individuals, and good reproducibility in the assay. In agreement with previous reports using EBV-transformed lymphoblasts, we show a very poor induction of apoptosis in AT homozygotes and a reduced level in AT heterozygotes compared to normal individuals. A similar reduced level compared to normal individuals was seen in the breast cancer patients. Despite a wide range of values in the breast cancer patients and good reproducibility on repeat samples, there was no correlation of rates of apoptosis with the severity of breast fibrosis, retraction or telangiectasia. The reduced rate of apoptosis observed in the breast cancer cases may be associated with genetic predisposition to breast cancer; however, we conclude that assays of lymphocyte apoptosis are unlikely to be of use in predicting normal tissue tolerance to radiotherapy.

Construction of a transcription map around the gene for ataxia telangiectasia: identification of at least four novel genes.

We have constructed YAC, PAC, and cosmid contigs in the ataxia-telangiectasia gene region and used the assembled clones to isolate expressed sequences by exon trapping and hybridization selection. In the interval between D11S1819 and D11S2029, exons and cDNAs for potentially 13 different genes were identified. Three of these genes, F37, K28, and 6.82, are large novel genes expressed in a variety of different tissues. K28 shows sequence homology to the Rab GTP binding protein family and gene 6.82 homology to the rabbit vasopressin activated calcium mobilizing receptor, while gene F37 has no homology to any known sequence in the database. Three further clones, exon 6.41 and cDNAs K22 and E74, from the interval between D11S1819 and D11S2029, appear to be expressed endogenous retrovirus sequences. The fourth large novel genes, E14, together with two further possible novel genes, E13 and E3, was identified from exons and cDNAs in the more telomeric 300-kb interval between markers D11S2029 and D11S2179. These are in addition to the genes for mitochondrial acetoacetyl-CoA-acetyltransferase (ACAT) and the ATM gene in the same region. Genes E3, E13, and E14 do not show homology to any known genes. K28, 6.82, ACAT, and ATM all appear to have the same transcriptional orientation toward the telomere.

The Saccharomyces cerevisiae MEC1 gene, which encodes a homolog of the human ATM gene product, is required for G1 arrest following radiation treatment.

The Saccharomyces cerevisiae gene MEC1 represents a structural homolog of the human gene ATM mutated in ataxia telangiectasia patients. Like human ataxia telangiectasia cell lines, mec1 mutants are defective in G2 and S-phase cell cycle checkpoints in response to radiation treatment. Here we show an additional defect in G1 arrest following treatment with UV light or gamma rays and map a defective arrest stage at or upstream of START in the yeast cell cycle.

Expression of the ATDC (ataxia telangiectasia group D-complementing) gene in A431 human squamous carcinoma cells.

The ATDC gene was originally identified by its ability to complement the radiosensitivity defect of an ataxia telangiectasia (AT) fibroblast cell line. Because hypersensitivity to ionizing radiation is an important feature of the AT phenotype, we reasoned that ATDC may function generally in the suppression of radiosensitivity. Previous work in our laboratory focused on radiosensitization mechanisms in human squamous carcinoma (SC) cells, especially A431 cells. To establish a basis for investigating the role of ATDC in radiation-responsive signaling pathways in human SC cells, we characterized ATDC message and protein expressions in A431 cells. ATDC message expression was also compared among human epidermoid cells (A431 cells, HaCaT spontaneously immortalized human keratinocytes and normal human epidermal keratinocytes) and a normal human fibroblast cell line (LM217). We made the following major observations: (i) the relative abundance of ATDC message is substantially higher in the epidermoid cells than in the fibroblast cell line, which has a message level comparable to those reported for other fibroblast lines; (ii) ATDC is constitutively phosphorylated on serine/threonine in A431 cells; (iii) in A431 cells, ATDC is a substrate for the serine/threonine protein kinase C (PKC) but not the epidermal growth factor (EGF) receptor tyrosine kinase; and (iv) EGF decreases ATDC message and protein expressions in A431 cells after a 24-hr exposure. The phosphorylation studies suggest that the ability of ATDC to modulate cellular radiosensitivity may be mediated in part through a PKC signaling pathway.

Nucleotide sequence analysis of a candidate gene for ataxia-telangiectasia group D (ATDC).

A radioresistant cell clone (1B3) was previously isolated after transfection of an ataxia-telangiectasia (AT) group D cell line with a human cosmid library. A cosmid rescued from the integration site in 1B3 contained human DNA from chromosome position 11q23, the same region shown by both genetic linkage and chromosome transfer to contain the genes for AT complementation groups A/B, C, and D. A gene within the cosmid (ATDC) was found to produce mRNAs of different sizes. A cDNA for one of the most abundant mRNAs (3.0 kb) was isolated from a HeLa cell library. In the present study, we sequenced the 3.0-kb cDNA and the surrounding intron DNA in the cosmids. We used polymerase chain reaction, with primers in the introns, to confirm the number of exons and to analyze DNA from AT group D cells for mutations within this gene. Although no mutations were found, we do not rule out the possibility that mutations may be present within the regulatory sequences or coding sequences found in other mRNAs specific for this gene. From the sequence analysis, we found that the ATDC gene product is one of a group of proteins that share multiple zinc finger motifs and an adjacent leucine zipper motif. These proteins have been proposed to form homo- or heterodimers involved in nucleic acid binding, consistent with the fact that many of these proteins appear to be transcriptional regulatory factors involved in carcinogenesis and/or differentiation. The likelihood that the ATDC gene product is involved in transcriptional regulation could explain the pleiomorphic characteristics of AT, including abnormal cell cycle regulation.