Ataxia telangiectasia (A-T) is a rare autosomal recessive disease characterized by progressive neurodegeneration and cerebellar ataxia. A-T is causally linked to defects in ATM, a master regulator of the response to and repair of DNA double-strand breaks. The molecular basis of cerebellar atrophy and neurodegeneration in A-T patients is unclear. Here we report and examine the significance of increased PARylation, low NAD + , and mitochondrial dysfunction in ATM-deficient neurons, mice, and worms. Treatments that replenish intracellular NAD + reduce the severity of A-T neuropathology, normalize neuromuscular function, delay memory loss, and extend lifespan in both animal models. Mechanistically, treatments that increase intracellular NAD + also stimulate neuronal DNA repair and improve mitochondrial quality via mitophagy. This work links two major theories on aging, DNA damage accumulation, and mitochondrial dysfunction through nuclear DNA damage-induced nuclear-mitochondrial signaling, and demonstrates that they are important pathophysiological determinants in premature aging of A-T, pointing to therapeutic interventions.

Here, we investigate MT dysfunction and its relationship to neurological defects in rat neurons, worms (C. elegans), and mouse models of A-T. Results presented here show that restoration of the NAD + /SIRT1 signaling reduces the severity of neurological defects and increases healthspan and lifespan in these model systems. NAD + replenishment also stimulates DNA repair and mitophagy. These results encourage consideration of therapeutic intervention in certain DNA repair-deficient diseases.

Defects in DNA repair can be associated with mitochondrial (MT) dysfunction, neuropathy, and ataxia, as reported in ATM (), Xeroderma pigmentosum complementation group A (XPA), and Cockayne syndrome group B (CSB) (). In these conditions, DNA repair defects are associated with persistent DNA damage, hyperactivation of poly-ADP-ribose polymerase 1 (PARP1), and low intracellular NAD, an essential co-factor for PARP1-catalyzed PARylation (PAR) of its target substrates (). NADis a key cellular factor linked to metabolism, MT health, stem cell rejuvenation, neuroprotection, and longevity in worms and mice (). NADis also a required co-factor of SIRT1, a nuclear deacetylase that modulates chromatin structure and regulates MT biogenesis by deacetylating PGC-1α, a nuclear transcription factor (). PARP1 and SIRT1 compete for NADand may reciprocally limit each other’s activity. Based on these and other observations reported by us and others, we propose that persistent DNA damage activates signaling from the nucleus to the mitochondria, which we call NM signaling. This DNA damage-induced NM signaling pathway may play an important role in normal aging and some specific age-related pathologies ().

Ataxia telangiectasia (A-T) is a rare autosomal recessive disease characterized by uncoordinated movement, telangiectasia, radiosensitivity, and cerebellar atrophy (). A-T is caused by mutation of the ataxia-telangiectasia mutated (ATM) gene, encoding the ATM kinase, a master regulator of DNA damage processing (). In response to DNA damage, the Mre11-Rad50-Nbs1 (MRN) complex is recruited to the site of the DNA double-strand breaks (DSBs), and then ATM is activated by autophosphorylation, which converts the inactive ATM dimer to an active monomer, promoting DNA repair (). ATM heterozygotes are common in the general human population (0.5%–1%), and this genotype is linked to elevated risk of cancer and some age-related diseases (). While some major features of A-T likely reflect defective repair of DSBs (DSBR), the molecular basis of cerebellar atrophy in A-T is poorly understood.

Encouraged by the healthspan improvements in mice ( Figure 7 ), we further investigated the effects of NADon the lifespan of the short-lived Atmmice. Both WT and Atmmice were given NR (12 mM) in drinking water at 1 month of age and continuously throughout their lifespans. NR treatment markedly extended the lifespan of Atmmice ( Figure 7 G). For the Atmvehicle group, approximately 50% of the mice died within 3.5 months, and all had passed by 5 months (102 ± 30.5 days in mean ± SD), consistent with previous reports (). Strikingly, nearly 80% of the NR-fed Atmmice survived over 10 months, indicating a dramatic improvement in lifespan by NADsupplementation. Extension of lifespan in Atmmice by NADsupplementation may be associated with improved mitophagy and DNA repair but also with the anticancer potential of NADor sirtuins (). Thymic lymphoblastic lymphomas are a major cause of death for Atmmice (). Analysis of the thymus tissues indicated that Atm(vehicle) tissues were heavier (0.125 g each) than that of WT (vehicle) littermates (0.035 g each) ( Figures S5 E and S5F). NR was able to reverse the hyper-proliferation of the thymus tissue; however, further analysis is ongoing to substantiate this observation. In summary, our results demonstrate that NR treatment normalizes MT function and reduces persistent DNA damage in brain tissue of Atmmice.

To test whether NR altered the level of endogenous DNA damage, comet assays were done on primary rat neurons that had been treated with scrambled small interfering RNA (siRNA) or with siATM. Loss of ATM increased endogenous levels of DNA DSBs that could be ameliorated by NR treatment ( Figure S6 K). This suggests that NR can reduce the steady level of endogenous DNA damage. Furthermore, consistent with the improved DNA repair observed in ATM-KD neurons and atm-1 worms after NR treatment ( Figure 5 ), both NR and NMN significantly decreased 53BP1 levels in Atmmice compared with vehicle, suggesting less accumulation of endogenous DNA breaks ( Figures 7 E and S6 I). Furthermore, a chemiluminescent PARP assay showed no difference in maximal PARP-1 activity between WT and Atmextracted PARP-1 protein ( Figure S6 J). Thus, NADrepletion decreased accumulation of endogenous DNA damage in ATM-KD neurons, likely through increased DNA repair via NHEJ.

Cerebellum tissues from Atmmice showed elevated PAR staining, increased acetylated p53, and greatly reduced levels of total CREB and p-CREB levels ( Figures 7 E and S6 A–S6C). We detected lower levels of cleaved caspase-3 in the NR- and NMN-treated Atmmice compared with vehicle control, suggesting less neuronal death ( Figures 7 E and S6 D). The protection by NR and NMN on Purkinje cell loss was confirmed by observing reduced levels of a specific Purkinje cell marker, calbindin, in the cerebellum of Atmmice compared to WT mice, and preservation of calbindin levels in Atmmice treated with NR or NMN ( Figures 7 E and S6 E). In Atmmice, NR and NMN treatment significantly increased LC3-II levels and promoted a redistribution of the fission-fusion balance by decreasing mitofusin-2 (an MT fusion protein), although there were no statistically significant changes on a fission marker, p-DRP1/DRP-1 ( Figures 7 E and S6 F–S6H). Consistently, large and damaged mitochondria were prominent in the Atmmouse cerebellum ( Figure 7 F). NR and NMN treatment normalized the MT morphology and minimized damaged mitochondria ( Figures 7 F, S5 B, and S5C). Additionally, liver and muscle tissues of Atmmice exhibited abnormal levels of several MT proteins that were normalized by NR and NMN treatment, indicating a likely systemic benefit of NADsupplementation on mitochondria ( Figure S5 D).

We further examined the effects of NADsupplementation by giving Atmmale mice and their age- and sex-matched WT littermates NR (12 mM) and another NADprecursor, NMN (12 mM) (), in their drinking water starting from weaning and continuing for 2 weeks. The NADlevels in the cerebellum of Atmmice were initially about 40% of those in WT, but after 14 days of treatment with NR or NMN, NADlevels increased significantly to levels similar to WT mice ( Figure 7 A). Atmmice had a significant deficit in motor function before any intervention and showed a dramatic improvement in the latency of time to their first fall from the rotarod after NR or NMN treatment ( Figure 7 B). While there was a significant benefit to Atmmice, there was no significant effect of NR or NMN on the performance of young WT mice. Memory was investigated using the Y maze spontaneous alternation behavioral test. Atmmice displayed roughly 50% of the exploratory behavior of WT mice, and, remarkably, both NR and NMN could restore this behavior to normal levels, suggesting improved memory functions ( Figure 7 C). Furthermore, the numbers of Purkinje cells in Atmmice treated with NR or NMN were significantly greater than vehicle-treated Atmmice, whereas NR and NMN had no significant effect on Purkinje cell numbers in WT mice ( Figures 7 D and S5 A).

(G) Kaplan-Meier survival curves of vehicle- and NR-treated mice. One-month-old Atm −/− mice and their age- and sex-matched WT littermates were exposed to NR (12 mM in drinking water; approximately 570–590 mg/kg/day/mouse) and lifespan was determined (12–20 mice/group). Statistical analysis was performed by the log-rank test.

(F) Representative EM images showing mitochondria in cerebellum tissues of Atmmice and their WT littermates after 14-day vehicle, NR, or NMN treatment (n = 3–5 mice/group). Damaged mitochondria were marked with red arrows. Quantification of data is in Figures S5 B and S5C.

(E) Immunoblot analysis of the indicated proteins from the cerebellum of Atmmice and their WT littermates after 14 days of treatment with vehicle, NR, or NMN. Quantification of data is in Figure S6

(D) Representative confocal microscopy images of the Purkinje cells in the cerebellum of Atmmice and their WT littermates after 14 days of NR or NMN treatment. Over ten cerebellar areas for each mouse were analyzed (n = 3–5 mice/group). Quantification of images is shown in Figure S5 A.

(A–C) Effects of 14 days of NR or NMN supplementation on the NAD + levels (A), rotarod performance (B), and spontaneous alternation performance (SAP) in the Y maze (C). Data shown are mean ± SEM (n = 3–5 mice/group).

NADplays an important role in the tricarboxylic acid (TCA) cycle, a set of reactions critical to energy metabolism. Metabolism-related proteins that are differentially expressed in atm-1 but restored to N2 levels by NR, SRT1720, or olaparib are listed in Figure 6 A. We also identified 156 metabolites in N2 and atm-1 worms ( Table S5 ). The metabolite profile in samples from nicotinamide mononucleotide (NMN)/NR-treated atm-1 worms clustered more closely with the metabolite profile of WT compared to atm-1 (vehicle) worms ( Figure 6 B). With regard to metabolites in the TCA cycle, atm-1 worms had lower citrate and isocitrate, but higher succinate, fumarate, and malate than N2 worms ( Figure 6 C). While the levels of ketogenic amino acids were higher in atm-1 worms, glucogenic amino acids did not show a consistent direction of change ( Figure 6 C). However, treatment of NR/NMN normalized many of these metabolites ( Figures 6 D–6G). Proteomic analysis gave results consistent with observed effects on metabolism ( Figures 6 H–6J). Parallel studies were conducted in Atmmice. Atmmice and their WT littermates were dosed with 12 mM NR in drinking water for 14 days starting at weaning. Metabolomics analysis on cerebellum tissues showed a total of 116 metabolites were identified ( Table S5 ). There was a distinct metabolite profile in Atmmice, and NR treatment normalized the metabolic profile of the Atmmice ( Figure 6 K). Collectively, the metabolomics analyses in worms and mice suggest that ATM deficiency induces major changes in the metabolome, and that treatment with NR normalizes the metabolic profile in atm-1 worms and in Atmmice.

(D–G) Changes of levels of citrate (D), isocitrate (E), fumarate (F), and lactate (G) in N2 and atm-1 with or without NR/NMN treatment.

(C) Changes of TCA cycle and catabolism of amino acids in atm-1 (vehicle) compared with N2 (vehicle). Amino acids in green are ketogenic, red are glucogenic, and dark ones are involved in both glucogenic and ketogenic processes.

(B) Unsupervised hierarchical clustering of day 6 worm metabolites among different groups. Whole worm tissues were subjected to ALEX-CIS GCTOF mass spectrometry with more than 156 metabolites identified ( Table S5 ).

(A) Proteomics data showing changes of metabolism-related proteins that fulfilled the “3C” standards. For 3C standards, see Figure 3 E. Each protein was ascribed a molecular function and is further described in Table S2

atm-1 worms display sensitivity to ionizing radiation (IR), low brood size (fecundity), and high male frequency (). In our studies, we observed that treatment with NR improved brood size by 40% ( Figure 5 D) and lowered male frequency from 6.6% to 0.8% ( Figure 5 E). The effect of NR on N2 and atm-1 worms exposed to IR (90 Gy) was examined in the late embryonic stage, when NHEJ is the only pathway for DSBR (). The results show that treatment with NR significantly improved atm-1 survival ( Figure 5 F; 41%–79.6% survival to L4). To confirm that this effect was dependent on NHEJ and not HR, two HR repair-deficient mutants (brc-1(tm1145) and brd-1(dw1)) and two NHEJ mutants (cku-70(tm1524) and cku-80(ok861)) were employed. While growth was not impaired in either brc-1 or brd-1 worms, cku70 and cku80 mutants were extremely sensitive to IR treatment, further confirming the specificity of the assay and the role of ATM in NHEJ in worms ( Figure 5 F). Taken together, our data indicate that in ATM-KD neurons and atm-1 worms, NHEJ repair and genomic instability can be improved by NR.

NHEJ repair was also measured using a reporter plasmid pEGFP-Pem1-Ad2 in ATM-KD and control neurons treated with or without NR (). Using this assay, ATM-KD neurons demonstrated 30% of the activity of control neurons ( Figure 5 C), but were upregulated by treatment with NR in a manner substantially dependent on SIRT1 ( Figure 5 C). Some residual effect of NR ( Figure 5 A, lane 10) is consistent with functional overlap between SIRT1 and SIRT6, the NADdependence of SIRT6, and its known roles in NHEJ and HR (). In line with this, in SIRT1 and SIRT6 double-KD mutants, no effects of NR were detected ( Figure 5 C). Collectively, these data suggest that NHEJ repair in neurons can be stimulated by NR in both scrambled and short interfering ATM (siATM)-treated cells, and further that the stimulation is dependent on SIRT1 and SIRT6.

ATM protein kinase plays an important role in homologous repair (HR)- and non-homologous end joining (NHEJ)-mediated DSBR, with NHEJ being the predominate DSBR pathway in neurons and other post-mitotic cells (). NHEJ-mediated DSBR requires DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a serine/threonine protein kinase in the same family as ATM (). During NHEJ-mediated DSBR, Ku70/Ku80 is recruited first to DSBs followed by DNA-PKcs, forming the DNA-PK complex (). Deacetylation of Ku70 by SIRT1 and auto-phosphorylation of DNA-PKcs at position S2056 are critical to activate DNA-PKcs (). In control and ATM-KD SH-SY5Y cells treated with or without etoposide and with or without NR ( Figure 5 A), ATM-KD SH-SY5Y cells had higher levels of acetyl-Ku70 than control cells, and this was normalized by treatment with NR ( Figure 5 A). Unstressed control cells had low auto-phosphorylation of DNA-PKcs, but this increased in etoposide-treated cells ( Figure 5 A). In ATM-KD SH-SY5Y cells, auto-phosphorylation of DNA-PKcs was also low and etoposide had little effect in untreated cells, but it increased in NR-treated cells ( Figure 5 A). This suggests a defect in NHEJ in ATM-KD SH-SY5Y cells that is partially corrected by treatment with NADrepletion. We further verified that NR-induced improvements in the DNA damage response signaling were dependent on SIRT1 by knocking down SIRT1 in the ATM-KD SH-SY5Y cells ( Figure 5 A). Of note, NR decreased the abundance of acetylated Ku70 in SIRT1-KD cells (lane 10, Figure 5 A), suggesting that a backup enzyme exists and provides a function redundant to SIRT1 in these cells. NR also causes an increase in Ku70 and DNA-PKcs on chromatin in cells with or without etoposide-induced DNA DSBs ( Figure 5 B).

(F) Embryonic NHEJ capacity was measured by scoring the percentage of late-stage embryos that could reach to the L4 stage 48 hr after irradiation with 90 Gy. Two homologous recombination mutants, brc-1(tm1145) and brd-1(dw1), were used as negative controls, while two NHEJ mutants, cku-70(tm1524) and cku-80(ok861), were positive controls. About 150–300 embryos/group were scored, and data shown are mean ± SEM.

(D and E) Effects of restoration of the NAD + /SIR-2.1 pathway on genomic instability revealed by brood size (D) and male frequency (E). The atm-1(gk186) worms were used for brood size counting (D), while late-generation atm-1(gk186);(h2681) worms were used for male frequency (E). Data are mean ± SEM (n = 20–30 worms/group, three replicates).

(C) NHEJ DNA repair efficiency was measured in neurons that were transfected with a pre-digested NHEJ reporter construct 3 days after either scrambled siRNA or siRNAs targeting the designated genes. Over 100 neurons/group were counted, and data shown are mean ± SEM.

(B) Representative immunoblots from control and ATM-KD SH-SY5Y cells showing that NAD + increases the accumulation of Ku70 and DNA-PKcs on chromatin. Soluble nuclear (S.N.) fractions and chromatin fractions were isolated by a commercial kit followed by detection of protein levels by immunoblot.

(A) Representative immunoblots from control and ATM-KD SH-SY5Y cells showing the effects of enhancement of the NAD + /SIRT1 pathway on deacetylation of Ku-70 and auto-phosphorylation of DNA-PKcs.

To verify the roles of daf-16 and dct-1 in the beneficial effects of NR on worm health, pharyngeal pumping rates of D7 worms were quantified in daf-16, pink-1, pdr-1, or dct-1 RNAi-treated N2 and atm-1 worms ( Figure 4 I). The beneficial effects of NR were significantly abrogated by KD of each of these four gene targets. Interestingly, we noticed some minor remaining benefits of NR in pink-1/pdr-1/daf-16/dct-1 RNAi-treated atm-1 worms, indicating possible participation of some NAD-related but mitophagy-independent pathways in promotion of healthspan in atm-1 worms. In summary, our data suggest NR improves healthspan at least in part by stimulating mitophagy.

Because SIR-2.1 can directly upregulate the transcriptional activity of DAF-16 (), we determined if sir-2.1 could affect dct-1 expression via daf-16. We knocked down sir-2.1 or daf-16 independently in N2 and found that dct-1 expression was dependent on SIR-2.1 ( Figure 4 H). Consistent with the lower DCT-1::GFP protein level in atm-1 ( Figure 4 G), dct-1 gene expression was only half of that in N2 worms. However, its expression could be robustly enhanced in atm-1 by supplementation with NR in an SIR-2.1- and DAF-16-dependent manner ( Figure 4 H). PINK-1 and PDR-1 are integral mitophagy mediators and are likely upstream of DCT-1 in the mitophagy pathway ( Figure 4 E) (). We further confirmed that the improved MT network morphology in atm-1 NR-treated worms could not be corrected after RNAi KD of dct-1 or pink-1, demonstrating that the positive effects of NR require these genes ( Figures S4 C and S4D).

It is well established that SIR-2.1 induces MT biogenesis in worms (), but its roles in mitophagy and the underlying molecular mechanisms are poorly understood. LGG-1 is the worm homolog of a key mammalian autophagy marker LC3, while DCT-1 is the worm homolog of NIX/BNIP3L, a protein that regulates mitophagy and has been implicated in longevity and healthspan (). The dct-1 gene can be regulated by the pro-longevity transcription factor DAF-16 (), but the role for SIR-2.1 in this pathway has not been investigated. We hypothesized that SIR-2.1 might regulate not only MT biogenesis but also mitophagy via the DCT-1 pathway ( Figure 4 E). We crossed atm-1 worms with an MT mitophagy reporter strain () to visualize the expression and co-localization of LGG-1 and DCT-1 in atm-1 worms ( Figure S4 A). The positive control, paraquat (), induced mitophagy in N2, but failed to induce mitophagy in atm-1, suggesting that ATM deficiency causes a defect in mitophagy ( Figures 4 F and S4 B). The co-localization coefficient of LGG1::DsRed and DCT-1::GFP proteins was substantially lower in atm-1 than N2 (∼50%), but NR treatment increased it in atm-1 to levels similar to N2 worms ( Figures 4 F and S4 B). The atm-1 worms treated with NR showed significantly elevated levels of DCT-1::GFP compared to vehicle-treated atm-1 worms ( Figure 4 G), demonstrating that NR can restore DCT-1 protein expression and improve mitophagy.

We investigated whether MT dysfunction associated with ATM-KD or mutation might be due to defective mitophagy, a process that degrades damaged mitochondria by a lysosome-mediated mechanism (). Imaging by EM revealed increased size heterogeneity, more damaged mitochondria, and less evidence of ongoing mitophagy in ATM-KD than in control SH-SY5Y cells ( Figures 4 A and 4B ), and the MT defects were significantly normalized by treated with NR ( Figures 4 A, 4B, and S3 A–S3D). Cells expressing a mitochondria-targeted form of monomeric Keima (mt-mKeima) were used to quantify mitophagy in ATM-KD cells (). Mitophagy is clearly suppressed in ATM-KD HeLa cells relative to WT. NR significantly stimulates mitophagy in ATM-KD cells and, to a lesser extent, also stimulates mitophagy in WT cells ( Figures 4 C and 4D). Consistently, defective mitophagy in ATM-KD neurons could be significantly rescued with NR as judged by co-localization of an MT marker cytochrome c oxidase subunit IV (COX-4) and LC3, a marker of autophagy ( Figures S3 E and S3F).

(I) Contribution of NR-induced mitophagy to the improvement of healthspan (pharyngeal pumping) in N2 and atm-1. Vehicle- or NR-supplemented worms were fed with control RNAi or RNAi targeting the designated mitophagy genes at the egg hatching stage, followed by analysis of pharyngeal pumping on D7 adult worms. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; n.s., not significant.

(F) Effects of NR (500 μM) on the induction of mitophagy in N2 and atm-1 worms. An MT toxicant, paraquat (1 mM), was used as a positive control. Data are the mean ± SEM (n = 37–57 muscle cells/group from 20 worms).

(C and D) Detection of mitophagy using an mt-mKeima fluorescent reporter in HeLa cells with or without NR treatment (500 μM). FCCP (30 μM for 3 hr) was used as positive control. Cells were imaged by confocal microscopy (C) and quantified for mitophagy (D).

(B) Quantification of MT length from images such as those shown in (A). Data are the mean ± SD (306–514 mitochondria in cells from 3 separate cultures).

We further looked for differentially regulated genes that are regulated by the neuroprotective CREB/CRH-1 (). In NR-treated D1 or D10 N2 worms, 7 or 209 CRH-1-regulated genes were differentially expressed, respectively ( Table S4 ). In contrast, in NR-treated D1 atm-1 worms, 137 or 219 CRH-1-regulated genes were differentially expressed, indicating that age-related changes are already evident in young atm-1 ( Table S4 ). In mammals, SIRT1 contributes to the stability of CREB (). The levels of the worm CREB homolog CRH-1 declined in an age-dependent manner and compound treatments could partially restore expression in D7 atm-1 ( Figure 2 J). Collectively, we have identified several pathways that NR, SRT1720, and olaparib altered to elicit a similar biological effect.

Two-sample t tests were used for pairwise comparison of normalized protein expression in the two strains and age groups in presence and absence of NR. The results indicate that age has a more significant effect on protein expression in atm-1 than N2 worms, and although some proteins were regulated in the same direction in D10 N2 and D10 atm-1, many expression changes appeared to be unique to atm-1 worms ( Figure 3 F; Table S3 ). Relatively few changes and no pathways were significantly altered by NR in D1 worms (data not shown). Although NR affected protein expression in N2 and atm-1 worms, the response was greater in atm-1 worms ( Figure 3 G; Table S3 ). The pathways affected by NR in D10 atm-1 worms were associated with growth, fertility, metabolism, and development ( Figures 3 H and S2 G).

Quantitative proteomic analyses were performed on four biological replicates from worms treated identically and in parallel to those used for gene expression analyses. A heatmap of the normalized protein expression data revealed that genotype and age were the most significant variables separating the datasets, and suggested that all the compounds are working through the same pathways ( Figure S2 F). Statistical approaches were used to analyze these data. First, we used the non-parametric Wilcoxon test across the entire dataset and identified 277 proteins that were differentially expressed in D10 atm-1 worms (D10 N2 as reference). They fall into the following Gene Ontology classifications: adult lifespan, locomotion, fertility, and reproduction ( Table S2 ). Three criteria (termed “3C,” listed in Figure 3 E) were used to identify proteins that are differentially expressed in atm-1 but restored to N2 levels by NR, SRT1720, or olaparib. This identified 99 proteins ( Table S2 ), of which 45 overlap with the 277 proteins from Wilcoxon’s test. Proteins affecting lifespan, locomotion, MT function and redox homeostasis, UPR (unfolded protein response), and DNA damage response were specifically altered by NR, SRT1720, or olaparib in D10 atm-1 animals ( Figure 3 E).

We used comparative microarray and proteomic analyses to elucidate potential molecular mechanisms. Animals were treated at L4 and harvested and analyzed at D1 (young) or D10 (old) ( Figure 3 A). NR, SRT1720, and olaparib induce/repress comparable numbers of genes to a similar extent in a similar direction in N2 and atm-1 worms ( Figures 3 A, S2 D, and S2E). Values on the lines connecting the spheres indicate upregulated genes in red and downregulated genes in blue. Pathway analysis identified 70 pathways affected by all three compounds in atm-1 worms: 54/70 pathways (77%) changed in the same direction for all three compounds ( Table S1 ), 49/70 pathways had a Z score ≥ | ± 1.5| for at least one compound, and 90% of these changed in the same direction ( Table S1 ). Venn diagrams of 49 pathways with Z score ≥ | ± 1.5| showed that 16 pathways were altered by all three compounds. These pathways included pharyngeal pumping, locomotion, genomic stability, and development ( Figures 3 B–3D). These data add to the identification of downstream molecular pathways impacted by NAD/SIR-2.1 signaling in atm-1 worms.

(E) The three protein selection criteria for NR/SRT/Ola-treated D10 atm-1 worms are shown along with a list of proteins that fulfilled these criteria. Each protein was ascribed a molecular function and is further described in Table S2

(B–D) There are 49 common pathways induced by all three compounds (NR, SRT, and Ola). For each pathway, at least one of the compounds induced a change of Z score ≥ | ± 1.5|. Venn diagram analysis of these pathways shows 16 shared pathways with 2 upregulated (B) and 14 downregulated (C), which are all listed in (D).

(A) Diagram showing unique and shared genes in N2 and atm-1 worms. For numbers, red denotes upregulated genes while blue denotes downregulated genes. Numbers on the line between the linked circles show shared numbers of genes. Acronyms for all 16 groups of treatments are denoted.

To further explore the underlying beneficial effects of NAD, we examined the expression levels of factors important for the MT stress response at three different ages of the worms. PAR increased steadily with age in N2 and atm-1 worms. The apparent steady-state level of PAR was higher in atm-1 than in N2 worms and the PAR level was lower in D7 and D17 atm-1 worms treated with NR, SRT1720, or olaparib ( Figure 2 J). However, PAR decreased less in D17 than in D7 worms treated with these agents, possibly because uptake decreases when worms stop eating at ∼D8 at 25°C. The worm homolog of CREB, CRH-1, was lower in atm-1 than in N2 worms at D7, but the level was normalized in D7 atm-1 worms treated with NR, SRT1720, or olaparib ( Figure 2 J). atm-1 worms also expressed HSP-6 at a lower level than N2 worms, and this marker was also normalized in D7 and D17 atm-1 worms treated with NR, SRT1720, or olaparib ( Figure 2 J). When similar experiments were carried out in atm-1(gk186);sir-2.1(ok434) worms, which lack expression of SIR-2.1, an ortholog to mammalian SIRT1, nearly all of the effects of NR, SRT1720, and olaparib noted above were blocked ( Figures 2 D and S2 C). These results indicate that atm-1 worms have defects in lifespan and healthspan that can be rescued by boosting the NAD/SIR-2.1 pathway.

We postulate that MT dysfunction contributes to neuropathology in A-T. Therefore, MT networks were visualized by confocal microscopy in atm-1 worms crossed with ccIs4251, a muscle-specific pmyo-3::gfp strain. In D4 N2 worms, the MT network was well organized in parallel to the myofilament lattice ( Figures 2 G and 2H) (). In contrast, the MT networks in D4 atm-1;ccIs4251 worms appeared disorganized and aggregated mitochondria were detected in ∼30% of the GFP-expressing cells. All three compounds normalized the MT network in atm-1 worms ( Figures 2 G and 2H). Electron microscopy (EM) showed that ≈15% of the sarcomeres were disrupted in atm-1 worms at D7 with thickening of cuticle and hypodermis ( Figure 2 I). This suggests that atm-1 worms exhibit features of premature aging.

Because ataxia is a major clinical finding in A-T patients (), we evaluated locomotion defects in atm-1 C. elegans. Adult atm-1 displayed lower swimming movements and rate of pharyngeal pumping, while treatment with NR, SRT1720, or olaparib improved these parameters in both atm-1 and N2 worms ( Figures 2 D and S2 C). As a measure of cognitive function, long- and short-term associated memory (LTAM and STAM, respectively) were measured using a chemotaxis assay () in atm-1 and N2 worms. To avoid confounding defects in locomotion and LTAM/STAM, these experiments were performed in D1 and D4 worms, before locomotion defects manifest themselves in atm-1 worms. Severe defects in LTAM and STAM were evident in D4, but not in D1, atm-1, in agreement with a progressive neuronal dysfunction. However, LTAM and STAM were normalized in worms treated with NR, SRT1720, or olaparib ( Figures 2 E and 2F).

Previous studies show that an allele of C. elegans ATM-1 that lacks the kinase domain, atm-1(gk186), hereafter called atm-1, confers an A-T-like phenotype, including genomic instability, sensitivity to radiation, and infertility (). atm-1 worms also have a significantly shorter lifespan (16.2 days) than N2 (WT) worms (19.1 days) ( Figures 2 A and S2 A), and lower intracellular NADthan N2 at adult day 7 (D7) ( Figure S2 B). As for ATM-KD neurons, we examined the effect of NR, SRT1720, or olaparib on worm health when treated from L4 stage (larval stage 4) onward. All three compounds increased median lifespan of atm-1 ( Figures 2 B, 2C, and S2 A). Combined treatment with NR and SRT1720 did not extend lifespan significantly beyond what was achieved with either treatment alone ( Figures 2 B, 2C, and S2 A), suggesting that they acted through overlapping pathways.

(J) Representative immunoblots from young (D1), middle aged (D10), and old (D17) N2 and atm-1 worms. For all the experiments, worms from L4 stage were exposed to vehicle control, NR (500 μM), SRT (10 μM), or Ola (500 nM).

(G and H) Effects of NR, SRT, and Ola on muscle MT network morphology of adult D4 N2 and atm-1 worms. A myo-3::gfp reporter gene was expressed in both nucleus and mitochondria to mark non-pharyngeal body wall muscle cells. Arbitrary score of MT network (mean ± SEM; n = 53–67 muscle cells from 20 worms) (G) and representative confocal images of each condition (H) are presented. MT morphology was scored on a scale from 1 to 5, where the value 1 denotes a severely impaired MT network and 5 indicates a very well-organized MT network.

(A–C) Lifespan of worms treated with vehicle (A), or NR, SRT, Ola, or NR+SRT beginning at the L4 stage in N2 (B) and atm-1 (C) worms (n = 150 worms).

Next, we determined if restoration of NADlevels and SIRT1 activity could reverse the MT phenotypes of ATM-KD cells. To replenish the NAD/SIRT1 signaling, we treated ATM-KD and WT neurons with the NADprecursor nicotinamide riboside (NR), SIRT1 activator SRT1720, and the PARP1 inhibitor olaparib. Both NR and olaparib treatment significantly increased NADlevels in control and ATM-KD neurons, whereas no significant effect was seen for SRT1720 ( Figure 1 E). Although SRT1720 did not increase NADlevels, it did increase SIRT1 activity ( Figures 1 D and S1 C), consistent with an allosteric activation of SIRT1 (). All three compounds ameliorated ATM loss-induced MT pathological phenotypes ( Figures 1 A–1C). NR also significantly decreased apoptosis in etoposide-treated cells and reduced staining with anti-53BP1, a marker for DNA damage ( Figures 1 F, 1G, and S1 D). All three compounds improved neurite morphology ( Figures 1 G and S1 E). At the protein level, the neurotrophic factor BDNF, the transcription factor CREB, and synapse markers Synapsin 1 and PSD95 were reduced in ATM-KD neurons ( Figure 1 H), consistent with a previous report (). Importantly, restoration of the NAD/SIRT1 pathway partially rescued the levels of these proteins ( Figure 1 H). Collectively, the results demonstrate that compromised NM signaling through the NAD/SIRT1 pathway contributes to the neuronal pathology in ATM-KD neurons by impairing MT function.

We investigated the effects of ATM knockdown (ATM-KD) on rat primary neurons and human SH-SY5Y neuroblastoma cells ( Figure S1 A, available online). ATM-KD cells displayed higher MT membrane potential, higher MT content, and more reactive oxygen species (ROS) than wild-type (WT) cells ( Figures 1 A–1C and S1 B). These features could reflect a low rate of ATP production, low expression of MT uncoupling proteins (), and/or inadequate/inefficient clearance of damaged mitochondria. In addition, ATM-KD neurons displayed increased PAR, suggesting increased activity of PARP1 ( Figures 1 D and S1 C). Consequently, these cells have low intracellular NAD Figure 1 E), as reported previously (). Consistent with competition between PARP1 and SIRT1 for NAD), p53 and PGC-1α are hyper-acetylated in these ATM-KD neurons, suggesting impaired deacetylation activity of SIRT1 ( Figures 1 D, S1 C, and S6 L).

(H) Levels of the indicated proteins were evaluated by immunoblot analysis in lysates of cultured control and ATM-KD neurons. Veh, vehicle (DMSO); NR, nicotinamide riboside, 500 μM; NMN, nicotinamide mononucleotide, 500 μM; Resv, resveratrol, 5 μM; SRT1720/SRT, 2 μM; Ola, olaparib, 500 nM; Veli, veliparib, 500 nM. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; n.s., not significant.

(D) Immunoblot of protein levels from control and ATM-KD neurons after various treatments. To detect acetylated PGC-1α, PGC-1α was immunoprecipitated, then blotted for acetyl lysine.

(A–C) Relative MT membrane potential (A), MT ROS (B), and MT content (C) in control and ATM-KD rat cortical neurons with different treatments. Values are the mean ± SEM (n = 6–10 cultures/group).

Discussion

This study links impaired deacetylation activity of SIRT1 to MT phenotypes of A-T and presents mechanistic insight into how the augmentation of NAD+ levels can be linked to MT health and neuroprotection. We investigated this network through the use of an SIRT1 activator, a PARP inhibitor, and NAD+ precursors. These three interventions improved ATM-deficient phenotypes and suggested shared pathways. Combining microarray and proteomics, we identified principal mechanisms likely underlying A-T phenotypes that can be pharmacologically reversed. For example, proteomic data suggest that pharyngeal pumping was affected in atm-1 worms, but not in N2. Behavioral studies verified that the pumping rate was decreased in atm-1 worms and improved through activation of the NAD+/SIRT1 pathway. Our key observations suggest that depletion of NAD+ may be a major mechanism of neurodegeneration in A-T. Supplementation of NAD+ rescued the transcriptomic, proteomic, and behavioral phenotypes of atm-1 worms and Atm−/− mice. Mechanistically, NAD+ replenishment increased DCT-1-associated mitophagy and improved NHEJ DNA repair through activation of Ku70 and DNA-PKcs.

Fang et al., 2016 Fang E.F.

Scheibye-Knudsen M.

Chua K.F.

Mattson M.P.

Croteau D.L.

Bohr V.A. Nuclear DNA damage signalling to mitochondria in ageing. + precursors improved A-T phenotypes through improvement of MT quality and mitophagy via reestablishment of SIRT1 activity. Furthermore, NAD+ replenishment increased DNA repair in ATM-KD neurons. NAD+-dependent SIRT1 participates in DNA repair through deacetylation and/or activation of major DSB repair proteins, including ATM and Ku70 ( Chalkiadaki and Guarente, 2015 Chalkiadaki A.

Guarente L. The multifaceted functions of sirtuins in cancer. Dobbin et al., 2013 Dobbin M.M.

Madabhushi R.

Pan L.

Chen Y.

Kim D.

Gao J.

Ahanonu B.

Pao P.C.

Qiu Y.

Zhao Y.

Tsai L.H. SIRT1 collaborates with ATM and HDAC1 to maintain genomic stability in neurons. Falck et al., 2005 Falck J.

Coates J.

Jackson S.P. Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage. Shiloh and Ziv, 2013 Shiloh Y.

Ziv Y. The ATM protein kinase: regulating the cellular response to genotoxic stress, and more. + supplementation can increase neuronal repair of DSBs through augmentation of the DNA-PKcs-regulated pathway. In addition to SIRT1, other NAD+-dependent sirtuin members, including SIRT6 and SIRT3, also likely contribute to the neuroprotection and DNA repair improvements by NAD+ supplementation seen here. SIRT6 functions in stabilization of DNA-PKcs on chromatin and enhances NHEJ ( Mao et al., 2011 Mao Z.

Hine C.

Tian X.

Van Meter M.

Au M.

Vaidya A.

Seluanov A.

Gorbunova V. SIRT6 promotes DNA repair under stress by activating PARP1. McCord et al., 2009 McCord R.A.

Michishita E.

Hong T.

Berber E.

Boxer L.D.

Kusumoto R.

Guan S.

Shi X.

Gozani O.

Burlingame A.L.

et al. SIRT6 stabilizes DNA-dependent protein kinase at chromatin for DNA double-strand break repair. + precursors ( Cantó et al., 2012 Cantó C.

Houtkooper R.H.

Pirinen E.

Youn D.Y.

Oosterveer M.H.

Cen Y.

Fernandez-Marcos P.J.

Yamamoto H.

Andreux P.A.

Cettour-Rose P.

et al. The NAD(+) precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity. Cheng et al., 2016 Cheng A.

Yang Y.

Zhou Y.

Maharana C.

Lu D.

Peng W.

Liu Y.

Wan R.

Marosi K.

Misiak M.

et al. Mitochondrial SIRT3 mediates adaptive responses of neurons to exercise and metabolic and excitatory challenges. mt after treatment with NAD+ precursors. Our data suggest interconnected responses involving DNA damage/repair, SIRT1 dysfunction, compromised mitophagy, and MT dysfunction in neurodegeneration of A-T. NAD+ replenishment works via a SIRT1-dependent mechanism, although there may also be SIRT1-independent mechanisms that improve lifespan and healthspan in A-T animal models. Our data suggest that impairment of multiple pathways contributes to the neurodegeneration of A-T. Although we cannot rule out the possibility that increased acetylation machinery in ATM-deficient cells contributes to the total acetylation level of proteins, our in vitro and in vivo data show compromised SIRT1 deacetylation activity in A-T, as evidenced by increased acetylation of its downstream proteins such as p53, PGC-1α, and Ku-70. Because cells with compromised mitophagy are susceptible to apoptotic death (), it is likely that mitophagy reduction contributes to neuronal death leading to neurodegeneration in A-T. Our data suggest two possible mechanisms of defective mitophagy in A-T, including the presence of longer/larger damaged mitochondria due to an imbalance of MT fission fusion and reduced expression of proteins required for mitophagy. NADprecursors improved A-T phenotypes through improvement of MT quality and mitophagy via reestablishment of SIRT1 activity. Furthermore, NADreplenishment increased DNA repair in ATM-KD neurons. NAD-dependent SIRT1 participates in DNA repair through deacetylation and/or activation of major DSB repair proteins, including ATM and Ku70 (). In line with overlapping functions between ATM and DNA-PKcs (), we show that NADsupplementation can increase neuronal repair of DSBs through augmentation of the DNA-PKcs-regulated pathway. In addition to SIRT1, other NAD-dependent sirtuin members, including SIRT6 and SIRT3, also likely contribute to the neuroprotection and DNA repair improvements by NADsupplementation seen here. SIRT6 functions in stabilization of DNA-PKcs on chromatin and enhances NHEJ (). SIRT3 is a major MT deacetylase and plays a significant role in maintaining neuronal survival under both endogenous and exogenous stresses and can be activated by NADprecursors (). We also observed induction of UPRafter treatment with NADprecursors. Our data suggest interconnected responses involving DNA damage/repair, SIRT1 dysfunction, compromised mitophagy, and MT dysfunction in neurodegeneration of A-T. NADreplenishment works via a SIRT1-dependent mechanism, although there may also be SIRT1-independent mechanisms that improve lifespan and healthspan in A-T animal models.

NAD+ replenishment caused a remarkable extension of lifespan in the Atm−/− mice. In addition to improved DNA repair and maintenance of MT homeostasis, NAD+-induced lifespan extension in the Atm−/− mice may also be due to multiple other mechanisms including elimination of ROS, improvement of immunity, and inhibition of thymic lymphomas. Notably, NAD+ supplementation significantly improved healthspan and lifespan in atm-1 worms, an organism that has no tumor growth, implicating major roles for MT quality maintenance and DNA repair in the therapeutic effects of NAD+ across species.

In summary, our results suggest that NAD+ depletion can induce severe neurodegeneration. Boosting NAD+ ameliorates neuropathological defects and markedly extends lifespan in an A-T mouse model. Our study further elucidates the molecular mechanisms involved in NAD+ regulation of mitophagy and enhancement of DNA repair in DNA repair deficiency. We describe how two classical hypotheses of aging converge to explain A-T symptoms, namely, MT dysfunction and persistent DNA damage in a cross-species study. While it remains to be seen how well NAD+ supplementation translates to the clinical treatment of A-T patients, our findings suggest novel therapeutic strategies to combat this devastating disease and possibly other premature aging disorders with DNA repair defects.