Dominant-negative Dmp53 extends life span through the dTOR pathway in D. melanogaster

Abstract

Expression of dominant-negative (DN) versions of the Drosophila ortholog of the tumor suppressor p53 extends fly life span in a Calorie Restriction (CR) dependent manner. DN-Dmp53 expression furthermore leads to reduction of Drosophila insulin-like peptide (dILP) 2 mRNA levels and a decrease in insulin/insulin-like growth factor-signaling activity (IIS) in the fly fat body. It is unclear by which mechanisms DN-Dmp53 extends longevity, and whether modulation of insulin-signaling activity plays a pivotal role in life span regulation by Dmp53. Here we show that life span extension due to DN-Dmp53 expression is likely due to reduction of Dmp53 activity and that decreased Dmp53 activity does not extend life span when dILP2 is concomitantly over expressed. Furthermore, extended longevity due to DN-Dmp53 expression does not further extend the life span of flies over expressing the IIS associated transcription factor dFoxO, indicating that DN-Dmp53-dependent life span extension may be related to IIS. However, reduction of dFoxO levels does not decrease DN-Dmp53-dependent longevity extension. Interestingly, when DN-Dmp53 is expressed in flies lacking the translation initiation controlling factor Thor/4E-BP, the downstream target of dTOR signaling, no increase in life span is observed. Taken together, these data suggest that Dmp53 may affect life span by differentially engaging the IIS and dTor pathways.

Figure 1. Life span extension by DN-Dmp53 is dependent on endogenous Dmp53

Survivorship curves of female ELAV-Switch-DN-Dmp53 flies are shown. (A) In a wt-Dmp53 background, expression of DN-Dmp53 (black) leads to extension of mean, median and maximum life span of 24%, 27% and 16%, respectively, over uninduced control flies (grey; for complete statistics, please see Table 1, line 2–7). (B) In a Dmp53 null background, induction of the DN-Dmp53 construct (black) does not lead to life span extension over uninduced controls (grey). (C and D) Two different independent experiments expressing DN-Dmp53 (black) in a Dmp53 null background also do not show life span extension over uninduced control (grey).

Figure 2. Longevity increase by DN-Dmp53 requires dILP2, but not dFoxO

Survivorship curves of female flies are shown (for complete statistical analysis, including p-values, mean, median and maximum life span, as well as replicate experiments, please refer to Table 1, lines 8–13, 24–31). (A) IPC-directed expression of DN-Dmp53 and simultaneous over expression of dILP2 using a dILP2 driver (black) fails to increase life span over that of control flies over expressing dILP2 only (grey). (B) Expression of DN-Dmp53 (black) or of dFoxO (red) using the combined ELAV-Switch/ S₁-32 driver leads to significant life span extension (statistical analysis can be found in Table 1). Expression of both dFoxO and DN-Dmp53 together using the combined drivers (green) does not lead to additive effects on longevity extension (all respective controls are in lighter colors). (C–D) Expression of two different DN-Dmp53 constructs using the ELAV-Switch driver leads to extended life spans (grey: controls; black: DN-Dmp53; panel C: Dmp53-H159N; panel D: Dmp53-R155H). This life span extension is still observed when the DN-Dmp53 constructs are expressed in the dFoxO deficient background dFoxO²¹ (light green: controls; green: DN-Dmp53).

Figure 3. 4E-BP is required, but not sufficient for DN-Dmp53-dependent longevity extension

Survivorship curves of female flies are shown (for complete statistics, please see Table 1, lines 14–17, 22). (A) Pan-neuronal adult expression of DN-Dmp53 using the ELAV-Switch driver extends life span in a 4E-BP wild-type background (grey: controls; black: DN-Dmp53; background: Thor^1RV, a precise P-element excision line of the P-element insertion Thor¹ line). (B) When 4E-BP is deleted, DN-Dmp53 fails to increase longevity (grey: controls; black: DN-Dmp53; background: Thor^1Δ, an imprecise P-element excision line of the P-element insertion Thor¹ line). (C) Over expression of 4E-BP in the adult peri-cerebral fat body using the S₁-32 driver marginally extends fly life span (grey: controls; black: 4E-BP).

Figure 4. 4E-BP is required for the longevity response under standard DR conditions

Mean life span as a function of food content. (A) Wild type flies (Thor^1RV) have shortened life span in the pathological zone (2% and 20%), but respond vigorously with a longevity increase when yeast extract content is reduced in the healthy zone from 15% to 5% (blue line). When sucrose content is varied (red line), mean life span varies around the 40 day mark with no significant increase when sucrose is reduced from 15% to 5%. (B) Flies missing 4E-BP (Thor^1Δ) do not respond to a decrease of yeast extract content from 15% to 5% in the food in the healthy food conditions zone with a significant increase in mean life span (green). There is an increase in life span in the pathological zone when yeast extract is reduced from 20% to 2% back to the mean life span of wt flies (shown are the averages of two independent experiments; the asterisks denote statistically significant mean life span differences).

Figure 5. A model for the longevity regulating pathways connected to Dmp53

Activity reduction of Dmp53 in the Drosophila insulin-producing cells leads to down regulation of dILP2 abundance by unknown mechanisms. Dmp53 activity can be reduced through deacetylation by dSir2, the activity of which is increased under CR/DR conditions. How dSir2 activity is increased by CR/DR is unclear, but it may be that the reduction of specific nutrient components, particularly essential amino acids (EAA), underlies the CR/DR effects. Reduced Dmp53 activity inhibits PI3K and Akt activity in the fly fat body. As a consequence, dFoxo is predominantly nuclear to control transcription, but this event does not control life span. The longevity response is transmitted through inhibition of the TOR pathway and the translational inhibitor 4E-BP. dFoxo may modify the overall magnitude of the 4E-BP response and provide feedback regulation of dILPs (the signaling events underlying CR/DR are in red). This model may also apply to mammalian systems, in which the relevant tissues and signaling pathways are conserved.