Life span extension and neuronal cell protection by Drosophila nicotinamidase

Abstract

The life span of model organisms can be modulated by environmental conditions that influence cellular metabolism, oxidation, or DNA integrity. The yeast nicotinamidase gene pnc1 was identified as a key transcriptional target and mediator of calorie restriction and stress-induced life span extension. PNC1 is thought to exert its effect on yeast life span by modulating cellular nicotinamide and NAD levels, resulting in increased activity of Sir2 family class III histone deacetylases. In Caenorhabditis elegans, knockdown of a pnc1 homolog was shown recently to shorten the worm life span, whereas its overexpression increased survival under conditions of oxidative stress. The function and regulation of nicotinamidases in higher organisms has not been determined. Here, we report the identification and biochemical characterization of the Drosophila nicotinamidase, D-NAAM, and demonstrate that its overexpression significantly increases median and maximal fly life span. The life span extension was reversed in Sir2 mutant flies, suggesting Sir2 dependence. Testing for physiological effectors of D-NAAM in Drosophila S2 cells, we identified oxidative stress as a primary regulator, both at the transcription level and protein activity. In contrast to the yeast model, stress factors such as high osmolarity and heat shock, calorie restriction, or inhibitors of TOR and phosphatidylinositol 3-kinase pathways do not appear to regulate D-NAAM in S2 cells. Interestingly, the expression of D-NAAM in human neuronal cells conferred protection from oxidative stress-induced cell death in a sirtuin-dependent manner. Together, our findings establish a life span extending the ability of nicotinamidase in flies and offer a role for nicotinamide-modulating genes in oxidative stress regulated pathways influencing longevity and neuronal cell survival.

FIGURE 1.

The Drosophila homolog of yeast PNC1 encodes an active nicotinamidase. A, comparison of the NAD⁺ salvage pathway between mammals and yeast/flies. The nomenclature used is as in Rongvaux et al. (9) and Revollo et al. (8): PNC1, nicotinamidase; Npt1, nicotinic acid phosphoribosyltransferase; Nma1&2, nicotinic acid mononucleotide adenylyltransferase 1 and 2; Qns1, NAD synthetase; Nmnat, nicotinic acid mononucleotide adenylyltransferase; NADS, NAD synthetase; NaMN, nicotinic acid mononucleotide; NMN, nicotinamide mononucleotide. D-NAAM is our designation for Drosophila nicotinamidase. B, Drosophila S2 cells were transfected with carboxyl-terminally tagged V5-His-D-NAAM using the indicated expression vectors, and D-NAAM expression was determined in whole cell lysates (TL) or in V5 immunoprecipitates by V5 immunoblotting. C and D, V5 immunoprecipitates from S2 cells expressing a control vector or pAc 5.1-V5-His-D-NAAM were assayed for amidase activity using nicotinamide (NAM, C) or pyrazinamide (PZA, D) as a substrate. The activity is provided in A₆₃₀ units corresponding to released ammonia. E, Drosophila S2 and mammalian COS-7 cells were transfected with control or pAc 5.1-V5-His-D-NAAM (S2 cells) or pExchange 5A-V5-His-D-NAAM (COS-7 cells) and nicotinamidase activity in V5 immunoprecipitates was analyzed as in C (top panel). D-NAAM recovery was determined using V5 immunoblotting (bottom panel).

FIGURE 2.

D-NAAM mRNA expression is regulated by oxidative stress. A, Drosophila S2 cells were cultured under the indicated growth conditions, and the expression of D-NAAM mRNA was analyzed using real time PCR. The data are presented as fold change in D-NAAM expression after standardizing to ribosomal protein L32. The treatments were as follows: C, control, complete SFM media; DS, DS2 media (media lacking protein factor additives); SFM 1:4, SFM medium diluted 1:4 with phosphate-buffered saline (nutrient deficient media), 18 h; DS 1:4, DS medium diluted 1:4 with phosphate-buffered saline, 18 h; FCS, 10% fetal calf serum, 4 h; LY, 10 μm LY294002, 18 h (PI3K inhibitor); HS1, heat shock, 2 h at 37 °C and 24 h recovery; HS2, heat shock, 2 h at 37 °C; H₂O₂, 15 μm, 18 h; Sor, 0.5 m sorbitol, 18 h; Ani, 10 μg/ml anisomycin, 18 h; DMSO, 0.1% Me₂SO, 18 h (control); Rapa, rapamycin, 0.2 or 0.5 μg/ml, 18 h; Res, 100 μm resveratrol, 18 h; NAM, 40 mm nicotinamide, 18 h; Sir, 50 μm sirtinol, 18 h. B, Drosophila S2 cells growing in complete SFM medium were treated as indicated or cells were irradiated and left to recover for 24 h. The cells were analyzed for changes in D-NAAM mRNA expression as in A. C, Drosophila S2 cells were treated with the indicated concentrations of H₂O₂ for the indicated times, and the D-NAAM mRNA levels were analyzed as in A.

FIGURE 3.

Oxidative stress enhances D-NAAM protein expression and activity. A, Drosophila S2 cells were cultured under the indicted growth condition (as in Fig. 2A) for 18 h, and total cell lysates were analyzed for D-NAAM protein expression using a carboxyl-terminal D-NAAM peptide antibody (top panel). Tubulin immunoblotting was used to confirm protein loading (bottom panel). Cell treatments and the protein expression analysis were performed in duplicate. DMSO, dimethyl sulfoxide. B, D-NAAM was immunopurified from S2 cells cultured under the indicated conditions using the carboxyl-terminal D-NAAM antibody and assayed for nicotinamidase activity as in Fig. 1. NC, negative control, no substrate (NAM) was added in the nicotinamidase assay. The experiment was performed in duplicate and is representative of three independent experiments. The treatments were as follows: C, control; HS, heat shock, 2 h at 37 °C; H₂O₂, 15 μm, 18 h; Ani, 10 μg/ml anisomycin, 18 h; DMSO, 0.1% Me₂SO, 18 h (control); Res, 100 μm resveratrol, 18 h; NAM, 40 mm nicotinamide, 18 h; Sir, 50 μm sirtinol, 18 h.

FIGURE 4.

Increased nicotinamidase activity in transgenic Drosophila lines overexpressing D-NAAM. A, expression of V5-D-NAAM protein in pUAST-D-NAAM transgenic lines in tubulin-Gal4 driver background was analyzed using V5 immunoblotting. B, D-NAAM protein expression in control w¹¹¹⁸ flies, a high expressing D-NAAM transgenic line 42am (tubulin-Gal4 driver), S2 cells, and control COS-7 cells were analyzed using a carboxyl-terminal D-NAAM antibody. C and D, nicotinamidase activity in protein extracts of the indicated fly lines or S2 cells was analyzed by immunoprecipitating (IP) D-NAAM using V5 or a carboxyl-terminal D-NAAM antibody and assaying amidase activity as in Fig. 2B. Preimmune serum for the D-NAAM antibody (PS) was used as a control antibody. The difference seen in activity between V5 and D-NAAM immunoprecipitates reflects the difference in immunoprecipitation efficiencies (data not shown). Note that the low activity seen in the w¹¹¹⁸ sample is a result of assay linearity limitations (the total amount of protein to be used for the immunoprecipitation was determined in a way to allow remaining in the linear range of the assay for the high expressing D-NAAM lines). NAM, nicotinamide.

FIGURE 5.

D-NAAM overexpression extends Drosophila life span. A-F, survival curves for male (A, C, and E) and female (B, D, and F) adult flies expressing low (D-NAAM³³; A and B) or middle (D-NAAM³¹; C and D) D-NAAM protein levels ubiquitously via the tubulin-Gal4 driver or of a high expressing line expressing D-NAAM strictly in neurons via the elav-Gal4 driver (D-NAAM⁴²; E and F; see Fig. 4A for D-NAAM expression levels in the specific lines). A genetically matching control line was used for each test line. Indicated are the n values for each experiment and the statistical parameter values. See supplemental Table S2 for the complete longevity analysis. G-J, mean (G and H) and the maximal life span of the top 20% survivors of test and control flies (I and J). The gray bars represent the percentage of change between the matching control and the test line for each pair. The single asterisk denotes a p value <0.005, and two asterisks denote a p value <0.05.

FIGURE 6.

D-NAAM-induced longevity requires Sir2. Survival curves of male (left panel) and female (right panel) adult flies expressing D-NAAM⁴² using the elav-Gal4 driver in control and Sir2 null background. Genotypes: wild type control: w¹¹¹⁸; D-NAAM overexpression: w¹¹¹⁸;UAS-D-NAAM⁴²/elav-Gal4; Sir2 null: w¹¹¹⁸; Sir2^4.5/Sir2^5.26; D-NAAM overexpression in Sir2 null: w¹¹¹⁸;Sir2^4.5/Sir2^5.26;UAS-D-NAAM⁴²/elav-Gal4; D-NAAM insertion control in Sir2 null: w¹¹¹⁸; Sir2^4.5/Sir2^5.26;UAS-D-NAAM⁴²/+; elav-Gal4 insertion control in Sir2 null: w¹¹¹⁸;Sir2^4.5/Sir2^5.26;elav-Gal4/+.

FIGURE 7.

D-NAAM expression protects human neuronal cells from oxidative stress-induced cell death. A-D, SH-SY5Y cells transfected with control vector or D-NAAM or nontransfected cells were exposed for 24 h to 100 or 300 μm NOC-9 and analyzed for cell death using trypan blue exclusion (A and B) or assayed for apoptotic cell death using deoxynucleotidyltransferase-mediated dUTP nick end labeling assay (C and D). For quantification of cell death and DNA fragmentation, on average, 200 cells were counted in triplicate samples (B and D). † denotes p value <0.01, and asterisks denote statistically nonsignificant difference between nontransfected and vector-transfected cells. E and F, control SH-SY5Y cells (E) or cells expressing EGFP control vector, EGFP-D-NAAM or EGFP-Sirt1 (F) were treated with the indicated concentrations of sirtinol (E) or 50 μm sirtinol (F) 1 h prior to exposure to 300 μm NOC-9 and cell survival was determined 24 h later as in B. G-J, SH-SY5Y cells were transfected with EGFP control vector, EGFP-D-NAAM, or EGFP-Sirt1; cell survival in response to 300 μm NOC-9 was analyzed as in B (G), and apoptotic cell death was analyzed as in D (I). Alternatively, EGFP positive and negative cells were analyzed separately (H and J). The asterisks denote p value <0.01.