Neuronal Expression of p53 Dominant-Negative Proteins in Adult Drosophila melanogaster Extends Life Span

Current Biology, Vol. 15, 2063–2068, November 22, 2005, ª2005 Elsevier Ltd All rights reserved.

DOI 10.1016/j.cub.2005.10.051

Neuronal Expression of p53 Dominant-Negative Proteins in Adult Drosophila melanogaster Extends Life Span Johannes H. Bauer,1,3 Peter C. Poon,1,3 Heather Glatt-Deeley,1 John M. Abrams,2 and Stephen L. Helfand1,3,* 1 University of Connecticut Health Center Department of Genetics and Developmental Biology 263 Farmington Avenue Farmington, Connecticut 06073 2 Department of Cell Biology University of Texas Southwestern Medical Center 5323 Harry Hines Boulevard Dallas, Texas 75390

Summary Hyperactivation of p53 leads to a reduction in tumor formation and an unexpected shortening of life span in two different model systems [1, 2]. The decreased life span occurs with signs of accelerated aging, such as osteoporosis, reduction in body weight, atrophy of organs, decreased stress resistance, and depletion of hematopoietic stem cells. These observations suggest a role for p53 in the determination of life span and the speculation that decreasing p53 activity may result in positive effects on some aging phenotypes [3, 4]. In this report, we show that expression of dominantnegative versions of Drosophila melanogaster p53 in adult neurons extends life span and increases genotoxic stress resistance in the fly. Consistent with this, a naturally occurring allele with decreased p53 activity has been associated with extended survival in humans [5]. Expression of the dominant-negative Drosophila melanogaster p53 constructs does not further increase the extended life span of flies that are calorie restricted, suggesting that a decrease in p53 activity may mediate a component of the calorie-restriction life span-extending pathway in flies.

Results and Discussion Expression of DN-Dmp53 in Neurons throughout Life Increases Adult Life Span The adult fruit fly, Drosophila melanogaster, is well suited for studying the effects of targeted p53 reduction. It consists of mostly postmitotic tissue, and its p53 homolog Dmp53 is well characterized [6]. Flies with no functional Dmp53 gene are viable, but sickly with a shortened life span (see Figure S1 in the Supplemental Data available with this article online), indicating that a global reduction in Dmp53 activity is detrimental. Since neuronal function has been implicated in life span determination in flies and nematodes [7], in order to determine whether a targeted reduction of Dmp53 activity would

*Correspondence: [email protected] 3 Present address: Department of Molecular Biology, Cell Biology, and Biochemistry, Division of Biology and Medicine, Laboratories for Molecular Medicine, Brown University, 70 Ship Street, Room 407, Providence, Rhode Island 02903.

prolong healthful adult life, we expressed two different altered forms of Dmp53 in neurons by using the tissuespecific UAS-GAL4 system [8]. The DN-Dmp53-Ct mutant is a C-terminal fragment, and the DN-Dmp53259H mutant carries a point mutation in the DNA binding domain. Even though structurally very different, both mutants form tetramers with endogenous Dmp53, but fail to bind DNA. Although we are presently unable to directly measure the affect of these dominant-negative Dmp53 constructs on p53 activity in adult neurons, both of these constructs are known to function as strong dominant-negative (DN) inhibitors of transcriptional activation by wild-type (wt)-Dmp53 in yeast, Drosophila S2 cell culture, and Drosophila embryos [9]. When expressed using the panneuronal ELAV driver, life span of the Dmp53-Ct mutant is extended by 58% in females and 32% in males (Figures 1A and 1B and Table 1). Overexpression of wt-Dmp53 in neurons during development is lethal (data not shown). Expression of DN-Dmp53 Specifically in Adult Neurons Extends Adult Life Span The control strain used for this experiment was w1118, the same strain in which both DN-Dmp53 constructs were generated. However, since genetic background effects often confound life span studies in Drosophila due to hybrid vigor effects, we expressed each of the DN-Dmp53 constructs in adult neurons by using the conditional GeneSwitch system (GeneSwitch-ELAV-GAL4) [10, 11]. This system eliminates genetic background effects since all flies share the same genetic background and only differ with respect to the presence of the inducing agent in the food. Furthermore, by inducing expression during adult life, it avoids potential confounding negative effects of DN-Dmp53 expression during development and can be used to determine the period in life during which reduction in neuronal Dmp53 activity is most beneficial. Expression of DN-Dmp53-Ct or DNDmp53-259H proteins specifically in adult neurons led to an 11%–26% extension in median life span and a 10%–16% increase in maximum life span (Figures 1C–1F). On standard cornmeal food, life span was extended in females expressing DN-Dmp53, while on a higher calorie diet [12], both males and females expressing DN-Dmp53 lived longer than genetically identical controls (Table 1). When wt-Dmp53 was overexpressed (Figure 1G) or lacZ expressed (Figure 1H) during adult life, no significant change in life span was observed. A variety of other proteins were also overexpressed, with no positive effect on life span (Table 1). Thus, neither the addition of RU486 in the food, nonspecific protein overexpression (including wt-p53), nor the activation of the GeneSwitch-GAL4 system itself is responsible for the life span extension. When DN-Dmp53 constructs are expressed in nonneuronal cell types with the RU486-inducible GeneSwitch system, including muscle and fat body, negative effects on life span are observed (Figure S1 and Table 1). These results are consistent with our finding that flies globally lacking Dmp53 activity have a shortened life span (Figure S1). These data indicate that apparent selective reduction of

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Figure 1. Expression of Dominant-Negative Dmp53 Constructs in Neurons Leads to Life Span Extension (A and B) Survivorship curves of female (A) and male (B) DN-Dmp53-Ct (black) and w1118 control (gray) flies crossed to C-155 ELAV driver flies. Females show average, median, and maximum life span extension of 51%, 58%, and 15% over controls, respectively (p < 0.0001, chi-square, 67.76). Males have extended life spans of 37%, 32%, and 5% over controls (p < 0.0001, chi-square, 22.85). (C–H) ELAV-GeneSwitch flies were crossed to the indicated UAS lines. (C) Survivorship curves of flies induced with RU486 to express the DN-Dmp53-Ct mutant (black) show 15%, 18%, and 16% increased average, median, and maximum lifespan, respectively, over uninduced flies (gray). Log rank analysis demonstrates significant differences between the two survivorship curves (p = 0.0001, chi-square, 15.11). (D) In another independent trial with the Dmp53-Ct mutant, life span extension of 18%, 26%, and 10% was observed (p < 0.0001, chi-square, 23.22). (E) Survivorship curves of flies induced with RU486 to express the DN-Dmp53-259H mutant (black) show 15%, 11%, and 10% increased average, median, and maximum lifespan, respectively, over uninduced flies (gray) (p < 0.0001, chi-square, 20.23). (F) A second trial with the Dmp53-259H mutant shows 14%, 13%, and 10% life span extension (p < 0.0001, chi-square, 32.12).

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Table 1. The Effect of Various Proteins Expressed in Different Expression Patterns on Life Span

Protein/Expression Pattern

Median LS (versus Control)

Median LS Extension

Max LS (versus Control)

Max LS Extension

c2

p Value

60/38 78/59

58% 32%

84/73 92/88

15% 5%

67.76 22.85

<0.0001 <0.0001

58/46 52/44 52/46 62/56 60/54

26% 18% 11% 13% 11%

90/82 80/69 82/75 78/71 73/66

10% 16% 10% 10% 11%

23.11 15.11 20.33 32.12 60.36

<0.0001 0.0001 <0.0001 <0.0001 <0.0001

52/46

13%

62/56

11%

14.3

0.0002

66/66

0%

83/76

9%

7.582

0.0059

68/70

23%

90/86

5%

0.5666

0.4516

66/64 63/61

3% 3%

82/82 85/87

0% 22%

2.891 1.081

0.0891 0.2985

44/52 42/44 74/68

215% 25% 28%

65/73 54/64 100/98

211% 216% 22%

28.08 12.25 1.756

<0.0001 0.0005 0.1851

50/54

28%

74/74

0%

0.1583

0.6907

25/38 44/48 40/50 52/52 50/50

234% 28% 220% 0% 0%

47/63 64/64 60/76 76/74 68/68

225% 0% 221% 3% 0%

115.2 0.02818 71.78 1.930 0.1237

<0.0001 0.8667 <0.0001 0.1648 0.7251

Constitutive Panneuronal Expression Dmp53-Ct/ELAV Females Dmp53-Ct/ELAV Males GeneSwitch Expression Panneuronal Dmp53-Ct Dmp53-Ct Dmp53-259H Dmp53-259H Dmp53-Ct (High-Cal Food, Females) Dmp53-Ct (High-Cal Food, Males) Dmp53-Ct (Low-Cal Food, Females) Dmp53-Ct (Low-Cal Food, Males) Dmp53 LacZ Other Tissues Dmp53-Ct, Muscle Dmp53-Ct, Fat Body, S1-106 Driver Dmp53-Ct, Fat Body, SH32 Driver Cell Cycle (Panneuronal) Cyclin B3 Apoptosis (Panneuronal) Grim Eiger DRONC DRONC-DN p35

The indicated UAS lines were crossed to the neuronal-specific ELAV driver, ELAV-GeneSwitch driver, muscle-specific MHC-GeneSwitch driver, or fat body drivers S1-106-GeneSwitch or SH32-GeneSwitch. Offspring were collected and life span was determined on standard cornmeal food (except where indicated) in the presence of the inducing agent RU486 or diluent in the food as per [12]. Only females are shown, except where indicated. Median and maximum life span, log rank analysis, percent change in median and maximum lifespan as compared to controls (w1118 for constitutive expression, without RU486 for GeneSwitch experiments), chi-square, and p values derived from survivorship curves of each protein and driver are shown.

Dmp53 activity in adult neurons is sufficient to extend life span in flies. All subsequent experiments were therefore carried out with DN-Dmp53 expression in adults only. DN-Dmp53-Expressing Long-Lived Flies Are Selectively Resistant to an Oxidative Stress Since p53 plays an important role in mediating the response to genotoxic damage, we hypothesized that flies expressing the DN-Dmp53 constructs might be more resistant to stressors that cause DNA damage. We investigated the resistance of the long-lived DN-Dmp53 flies to paraquat, a reactive oxygen species generator known to cause genotoxic damage, and we found that flies expressing DN-Dmp53 are more resistant to paraquat poisoning than control flies (Figure 2A). At the same time, flies expressing DN-Dmp53 in the muscle or the fat body were no more resistant to paraquat poisoning than control flies (data not shown). Generalized resistance to

stress is a feature of a number of long-lived mutants in both Drosophila [13] and C. elegans [14]. We therefore examined the resistance of the DN-Dmp53-expressing flies to starvation or heat. These are both stresses that do not selectively induce genotoxic damage [15]. As opposed to the increase in resistance to paraquat, the DNDmp53-expressing flies were no more resistant than control flies to starvation (Figure 2B) or heat stress (data not shown). Thus, apparent reduction in Dmp53 activity selectively confers increased resistance to stress associated with genotoxic damage. DN-Dmp53 Long-Lived Flies Have Normal Female Fecundity and Normal Spontaneous Physical Activity Reduction of female fecundity is known to be associated with an increase in life span in Drosophila [12, 16]. We therefore examined whether a reduction in fertility might

(G and H) In contrast, neither Dmp53-overexpressing flies (G) nor lacZ-overexpressing flies (H) show any difference in life span between RU486induced (black) and uninduced (gray) lines. Representative survivorship curves are shown for female flies only. Male life span for DN-Dmp53-Ct adult neuronal-specific expressing flies was extended on high-calorie food (see Table 1 and Figure 3). RT-PCR and Q-PCR demonstrated a >40fold increase in expression of the Dmp53 constructs (wt-Dmp53, Dmp53-Ct, Dmp53-259H).

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Figure 2. Expression of Dominant-Negative Dmp53 Constructs in Adult Neurons Selectively Leads to Resistance to Paraquat with No Reduction in Fertility or Physical Activity Long-lived female ELAV-GeneSwitch/UASDN-Dmp53-Ct flies raised on standard cornmeal food were tested for physiological phenotypes associated with longevity. (A) A representative survival curve of flies exposed to 20 mM paraquat. Flies induced to express the Ct mutant (black) in adult neurons survive longer than uninduced flies (gray). Error bars represent the standard deviation. (B) Survivorship curves of flies exposed to starvation conditions. No difference in survival is seen between uninduced (gray) and induced (black) DN-Dmp53-Ct flies. This curve is a composite of two independent experiments; error bars represent 95% confidence intervals. (C) Daily egg production for ELAV-GeneSwitch/ UAS-DN-Dmp53-Ct flies over a period of 40 days. No statistically significant difference in daily egg production was observed between induced (black) and uninduced (gray) flies. Error bars are omitted for clarity. Lifetime egg production was similar (data not shown). (D) Spontaneous physical activity during a 24 hr period for ELAV-GeneSwitch/UAS-DN-Dmp53-Ct flies was measured at the indicated ages. No significant differences in physical activity were observed between uninduced (white) and induced (black) flies. Error bars represent the standard deviation. Similar results were also obtained when the DN-Dmp53-259H mutant was used (Figure S2).

account for the observed life span extension in females. No difference was observed in daily egg production over a period of 40 days (Figure 2C). Life span extension can be achieved through tradeoffs in physiological functions [17]. Therefore, we investigated whether diminished spontaneous physical activity accompanied extended life span in animals expressing DN-Dmp53. We found no difference in either the total or average amount of spontaneous activity between the uninduced control and the long-lived DN-Dmp53expressing flies at any age (Figure 2D). Therefore, it appears that no major physiological tradeoffs, neither a reduction in egg production nor physical activity, are contributing factors to the life span extension seen in flies with apparent specific neuronal reduction in Dmp53 activity. DN-Dmp53 Expression Does Not Further Extend the Life Span of Calorie-Restricted Long-Lived Flies Another method known to extend life span in a variety of species, spanning mammals, flies, nematodes, and yeast, is calorie restriction (CR) We therefore tested DN-Dmp53-overexpressing flies on food known to elicit a strong CR response [12, 18]. The low-calorie food (5% SY, see Supplemental Experimental Procedures) lies near the optimum on the calorie/life span scale for the strain of flies we are using [19], while flies on high-calorie food (15% SY) show life spans similar to flies on a cornmeal diet. In the fly strains used in this study, the life span of uninduced female control flies on low-calorie food (0.5N) is increased by 22% compared to control flies raised on higher calorie food (1.5N). This life span extension is not further increased when DN-Dmp53 is also expressed (Figure 3). The increase in life span for DN-Dmp53-expressing flies on 1.5N food as opposed to 0.5N food is unlikely to be due to an increase in uptake of food/RU486, since we find that female flies on 1.5N

and 0.5N take in an equivalent amount of food while males take in slightly more food on the lower 0.5N diet than on the higher 1.5N diet [19]. Lack of an additive effect on life span extension by combining CR and DNDmp53 expression in adult neurons suggests that both interventions may involve a similar pathway. Increasing Antiapoptotic Factors in the Adult Nervous System Does Not Increase Life Span Since p53 is known to be involved in controlling stressinduced apoptosis, a reduction in p53 might extend life span by decreasing apoptosis of irreplaceable postmitotic cells [3]. To determine what role reducing apoptosis in adult neurons may have on life span in the fly, we investigated whether targeted antiapoptotic interventions could also positively affect life span. Expression of antiapoptotic genes (baculovirus pancaspase inhibitor p35, DN-DRONC, dIAP1) at comparable levels to DN-Dmp53 (as evidenced by RT-PCR, not shown) via the same neuronal-specific GeneSwitch driver system did not lead to life span extension (Table 1). Proapoptotic proteins like Grim, Reaper, or the caspase DRONC, as expected, shortened life span. Expression of cell-cycle regulators had no effects on life span. Though expression of dominant-negative Dmp53 constructs specifically in adult neurons extends life span in D. melanogaster, our data suggest that this is unlikely to be due to a decrease in adult neuronal apoptosis, unless it is through a caspase-independent apoptotic pathway [20]. The role of p53 in responding to genomic damage is thought to be important in suppressing the emergence of tumors in mammals. Mice with increased expression of the 24 kDa or 44 kDa forms of p53 are resistant to tumor formation, but unexpectedly have a shortened life span with premature development of a number of phenotypes associated with aging [1, 2]. The activation of

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Figure 3. Life Span Extension of Flies Expressing Dominant-Negative Dmp53 Constructs in Adult Neurons Is Related to the CR Pathway of Life Span Extension Survivorship curves of ELAV-GeneSwitch/UAS-DN-Dmp53-Ct flies expressing DN-Dmp53-Ct on low-calorie and high-calorie food as compared to uninduced control flies. Both male and female flies induced to express DN-Dmp53 demonstrate life span extension over uninduced flies when raised on high-calorie food (statistical analysis in Table 1). The typical calorie-restriction life span extension can be seen by comparing uninduced flies on high-calorie to uninduced flies on low-calorie food (22% median; 15% maximum life span extension). However, no further increase in life span is observed when long-lived flies on low-calorie food are induced to express DN-Dmp53 (Table 1).

p53, in addition to suppressing tumor formation, may thus contribute to a variety of aging phenotypes. We found that increased expression of wt-Dmp53 in neurons during development is developmentally lethal in the fly (data not shown), but restricting overexpression to adult neurons did not shorten life span (Figure 1). Drosophila is an organism that is not prone to tumor formation yet possesses a p53-like molecule, Dmp53 [9, 21, 22]. The activity of Dmp53 during development may be important to remove or inactivate damaged cells before they go on to divide, form organs, and cause extensive dysfunction. Larvae lacking Dmp53 when subjected to radiation have a greatly reduced ability to form a viable adult fly [23]. Our studies support a positive role for Dmp53 during development and/or in some adult tissues since Dmp53 null flies or flies expressing dominant-negative Dmp53 constructs, specifically in adult muscle or fat body cells, have a shortened life span. Conversely, it has been suggested that a selective decrease in p53 activity might attenuate some of the negative effects of p53 and allow for an increase in healthy life span [3, 24]. In the adult fly, which is primarily a postmitotic organism, activation of a mechanism for removing or inactivating cells that cannot be replaced may have disadvantages. Indeed, we found that an apparent decrease in Dmp53 activity in neuronal tissue throughout life or restricted to adult neurons, via the expression of two very different, well-characterized dominant-negative proteins, DN-Dmp53-Ct or DN-Dmp53-259H, extended life span without significant physiological tradeoffs in reproduction or spontaneous physical activity.

Examination of the molecular and cellular mechanisms by which an apparent reduction in neuronal Dmp53 activity extends life span failed to show a relationship between the expression of antiapoptotic factors in the adult nervous system and life span extension. Apoptosis specifically associated with the adult nervous system has not been shown to play a prominent role in fly aging [25]. Suppression of the standard apoptotic pathways in adult neurons via decreased neuronal Dmp53 activity may not be a principle mediator of DNDmp53-dependent life span extension. Although the specific molecular and cellular mechanisms by which an apparent reduction in Dmp53 activity extends life span are not yet known, our studies demonstrating that expression of dominant-negative Dmp53 constructs specifically in adult neurons cannot further extend the life span of flies subjected to CR indicate that the life span extension of the DN-Dmp53-expressing flies may be related to the CR life span-extending pathway. In flies, the CR life span-extending effect has been linked to dSir2 expression, since a reduction in dSir2 blocks the CR life span-extending effect and the life span-extending effect of overexpressing dSir2 in neurons is not additive with CR [12]. Thus, both the life span extension mediated by an increase in neuronal dSir2 or neuronal expression of dominant-negative Dmp53 appear to be associated with the CR life spanextending pathway. Since Sir2 is known to inhibit p53 activity in mammalian systems, this raises the possibility that in flies, a decrease in Dmp53 may be one of the mediators of the CR/Sir life span-extending pathway.

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In this report, DN-Dmp53 was expressed in neurons, a tissue with reduced tumorigenic potential in adult invertebrates and humans. That p53’s antitumorigenic role should interact negatively with longevity, through an as yet uncharacterized mechanism, is intriguing. Nevertheless, conservation of p53 function across species suggests that neuronally targeted reduction of p53 should increase life span in other species in which global deactivation of p53 would otherwise lead to tumor formation and suggests a point of entry for the development of therapeutic agents to extend healthy life span. Supplemental Data Supplemental Data include two figures and Supplemental Experimental Procedures and can be found with this article online at http:// www.current-biology.com/cgi/content/full/15/22/2063/DC1/. Acknowledgements The authors would like to thank R.A. Reenan and J. Jack for critical reading of the manuscript and H. Keshishian, R. Davis, K. Basler, M. Tatar, and the Drosophila Stock Center (Bloomington) for fly stocks. We thank S. Kowalski, S. Goupil, and A. Sanchez-Blanco for technical assistance. This work was supported by grants from the NIA (AG16667, AG24353), the Donaghue Foundation, and the Ellison Medical Foundation to S.L.H. S.L.H. is an Ellison Medical Research Foundation Senior Investigator. This research was conducted while J.H.B. was a Glenn/AFAR Postdoctoral Fellow. Received: August 1, 2005 Revised: October 6, 2005 Accepted: October 10, 2005 Published: November 21, 2005

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