Results and discussion
Serotonin (5-HT) is a dose-sensitive neurotransmitter that modulates a variety of biological processes and behaviors. In the fruit fly, a quantitative trait locus for the variation of longevity has been mapped in the aromatic l-amino acid decarboxylase (Ddc) gene, which is required for serotonin and dopamine biosynthesis (De Luca et al., 2003). In mammals, age-related changes in serotonin signal are known risk factors of age-related diseases, such as diabetes and cardiovascular diseases, in which serotonin receptors, 5-HT1A and 5-HT2C receptors, may play a role (Mattson et al., 2004). These observations in the fruit fly and in mammals prompted us to test the hypothesis that serotonin signal may modulate longevity in Caenorhabditis elegans.
We investigated mutations that alter levels of serotonin and/or dopamine. They include mutations in serotonin biosynthesis genes (tph-1 and bas-1; reducing serotonin), a mutation in a dopamine biosynthesis gene (cat-2; reducing dopamine), a double mutation in dopamine and serotonin biosynthesis genes (bas-1;cat-4; reducing both dopamine and serotonin), and a mutation in a serotonin transporter gene (mod-5; increasing serotonin availability) (Loer & Kenyon, 1993; Ranganathan et al., 2000, 2001; Sawin et al., 2000; Sze et al., 2000; Dempsey et al., 2005). The mod-5 mutation causes effects similar to serotonin-reuptake inhibitors that are used as antidepressants (Ranganathan et al., 2001). All the genes used in this study and their orthologues are shown in Table 1.
|C. elegans||Fruit fly||Mouse||Human|
|bas-1||Ddc||Ddc||DDC||Aromatic l-amino acid decarboxylase (DOPA decarboxylase)|
|cat-4||Pu||Gch1||GCH1||GTP cyclohydrolase I|
|daf-2||InR||Insr, Igf1r, Insrr||INSR, IGF-1R, INSRR||Insulin/IGF-1 receptor|
|daf-16||foxo (dFOXO)||Foxo1, Foxo3a, Mllt7 (Foxo4)||FOXO1, FOXO3a, FOXO4||FOXO/forkhead transcription factor|
|mod-1||Rdl*||Gabrb3*||GABRB3*||Unique serotonin-gated ion channel similar to mammalian GABA- and glycine-gated chloride channels|
|mod-5||SerT||Slc6a4||SLC6A4||Na+, Cl−-dependent serotonin transporter|
|ser-1||5-HT1A†, 5-HT2†||Htr2a, Htr2c||HTR2A, HTR2C||5-HT2 receptor‡|
|ser-4||5-HT1A, 5-HT1B||Htr1a, Htr1b||HTR1B, HTR1D, HTR1A, HTR1F||5-HT1 receptor‡|
|tph-1||Trh||Tph1, Tph2||TPH1, TPH2||Tryptophan hydroxylase|
The results are summarized in Fig. 1 and Table 2. The serotonin biosynthesis mutations (tph-1 and bas-1) and the dopamine biosynthesis mutation (cat-2) caused no effect on lifespan (Fig. 1a,b). Thus, it is unlikely that reducing serotonin biosynthesis has an effect on lifespan under normal growth conditions. The tph-1 mutant had little effect on lifespan, but showed a prolonged reproduction period (P < 0.0001): Mean reproductive spans were: 4.4 ± 0.2 days for N2 (n = 23); 3.2 ± 0.0 days for ser-1(ok345) (n = 26); 5.1 ± 0.0 days for tph-1(mg280) (n= 17). The result of reproduction span in the tph-1 mutant is consistent with the previous study (Sze et al., 2000). The results suggest that there is an uncoupling between lifespan and reproductive lifespan, and it has been reported that there is such an uncoupling in many life-extending mutants (e.g. Murakami & Johnson, 1996; Gems et al., 1998; Jenkins et al., 2004). Interestingly, the tph-1 mutant modestly extended lifespan in the presence of FUDR (5-Fluoro-2′-deoxyuridine) (see below). In contrast, the mod-5 mutation caused shortened lifespan (P = 0.037; Fig. 1c). There was little difference in the survival curve of the mod-5 mutant during early mid phase of aging (day 2–8; before the end of reproduction). This result suggests that increasing the availability of serotonin may cause life-shortening. Taken together, reducing serotonin biosynthesis has little or modest effect on lifespan, while increasing serotonin may have a modest life-shortening effect. Note that the cat-2 mutant with reduced dopamine showed no effect on lifespan, while the bas-1; cat-4 double mutant with reduced dopamine and serotonin showed short lifespan (Fig. 1b).
|Genotype||Mean lifespan (days)||% change†||Maximum lifespan (days)||No. of replications‡||n||Comment|
|wild-type||11.5 ± 0.3||–||19.1 ± 1.3||8||175|
|bas-1(ad466)||10.8 ± 0.2||−6.1||17.3 ± 0.7||4||90||Serotonin biosynthesis|
|cat-2(e1112)||11.4 ± 0.3||−0.9||23.3 ± 2.1||4||96||Dopamine biosynthesis|
|bas-1(ad466); cat-4(e1141)||9.9 ± 0.9||−14.0||14.3 ± 0.7||4||90||Dopamine and serotonin biosynthesis|
|mod-1(ok103)||11.9 ± 0.3||3.5||25.2 ± 0.2||4||101||Serotonin-gated chloride channel|
|mod-5(n822)||10.7 ± 0.3||−7.0||19.5 ± 2.3||4||89||Serotonin reuptake|
|ser-1(ok345)||15.4 ± 0.4§||34.0||27.0 ± 1.2||4||88||Serotonin receptor|
|ser-1(ok345) + p(ser-1)||12.0 ± 0.4||4.3||22.0 ± 1.7||2||44|
|ser-4(ok512)||10.2 ± 0.3||−11.3||21.3 ± 2.1||4||90||Serotonin receptor P = 0.01 (day0–15)|
|tph-1(mg280)||11.4 ± 0.4||−0.9||23.3 ± 1.5||4||99||Serotonin biosynthesis|
|wild-type*||12.0 ± 0.4||–||17.6 ± 0.3||4||100|
|ser-1(ok345)*||14.9 ± 0.4§||24.2||22.5 ± 0.9||4||100|
|daf-2(RNAi)||14.3 ± 0.4§||19.2||22.5 ± 0.9||4||100||Insulin/IGF-1 receptor|
|ser-1(ok345); daf-2(RNAi)||14.8 ± 0.4§||23.3||22.0 ± 1.0||4||100|
|wild-type*||11.3 ± 0.3||–||17.4 ± 0.2||8||179|
|ser-1(ok345)*||15.9 ± 0.5§||40.7||21.8 ± 0.6||8||181|
|daf-16(RNAi)||11.1 ± 0.2||−1.8||14.5 ± 0.4||8||163||Forkhead transcription factor|
|ser-1(ok345); daf-16(RNAi)||11.5 ± 0.2||1.8||14.6 ± 0.4||8||179|
|wild-type*||12.0 ± 0.5§||–||20.0 ± 1.1||4||79|
|eat-2(ad465)*||16.0 ± 0.7§||33.3||24.0 ± 1.7||4||79|
|ser-1(RNAi)||17.5 ± 0.5§||45.8||29.3 ± 1.1||4||84|
|ser-1(RNAi); eat-2(ad465)||20.9 ± 1.1§||74.2||38.5 ± 3.1||4||81|
|mod-5(n822)*||9.5 ± 0.3§||−20.8||17.8 ± 0.3||4||80|
|ser-1(RNAi); mod-5(n822)||12.2 ± 0.1||1.7||22.0 ± 1.2||4||81|
|Treated with 1 mg mL−1 serotonin|
|wild-type-FUDR||15.5 ± 0.4||–||22.8 ± 0.8||4||79|
|wild-type-FUDR + serotonin||14.7 ± 0.5||−5.2||20.0 ± 0.7||4||81|
|tph-1(mg280)-FUDR||18.6 ± 0.4¶||20.0||28.8 ± 1.4||4||71|
|tph-1(mg280)-FUDR + serotonin||17.1 ± 0.3¶||10.3||22.8 ± 0.9||4||68|
|ser-1(ok345)-FUDR||17.7 ± 1.2§||14.2||31.8 ± 0.6||4||74|
|ser-1(ok345)-FUDR + serotonin||16.1 ± 0.5||3.9||26.5 ± 0.3||4||74|
|Treated with 5 mg mL−1 serotonin|
|wild-type-FUDR||14.6 ± 0.7||–||18.0 ± 0.6||4||77|
|wild-type-FUDR + serotonin||14.8 ± 1.0||1.4||18.5 ± 0.5||4||79|
|tph-1(mg280)-FUDR||16.7 ± 0.4§||14.1||24.0 ± 0.0||4||74|
|tph-1(mg280)-FUDR + serotonin||13.6 ± 0.7||−6.8||20.5 ± 0.5||4||77|
|ser-1(ok345)-FUDR||16.8 ± 1.6§||15.1||24.5 ± 1.7||4||79|
|ser-1(ok345)-FUDR + serotonin||17.8 ± 0.9§||21.9||24.0 ± 0.0||4||61|
We further investigated the hypothesis that serotonin influences lifespan at levels of downstream receptors. To this end, we tested mutants in three serotonin receptor genes, including two seven-transmembrane G-protein-coupled receptors for serotonin (ser-1 and ser-4) and a serotonin-gated chloride channel gene (mod-1) (Table 1). It has been shown that SER-1 responds to serotonin but not to other biogenic amines, including dopamine, histamine, and tryptamine (Hadman et al., 1999). SER-1 has properties of both 5-HT1 receptor (5-HT1R) and 5-HT2 receptor (5-HT2R). By the pharmacological definition, SER-1 is 5-HT1R (Hadman et al., 1999; Dempsey et al., 2005), while by the protein similarity, SER-1 is closely related to 5-HT2R (Hadman et al., 1999; http://www.wormbase.org). Similarly, SER-4 has the pharmacological property of 5-HT1R and is structurally similar to 5-HT2R (Demsey et al., 2005; WormBase; http://www.wormbase.org). Interestingly, a BLAST search identified the fruit fly dopamine receptors (DopR2 and D2R) and serotonin receptors (5-HT1A and 5-HT2) that are structurally similar to SER-1 (http://www.wormbase.org; data not shown). Because SER-1 has an activity as a serotonin receptor but not as a dopamine receptor (Hadman et al., 1999), it is likely that the dopamine receptor genes are structurally similar but functionally different. Note that the MOD-1 serotonin chloride channel is unique to C. elegans, and, thus, an example of similar genes is shown in Table 1.
The three serotonin-receptor mutations had different effects on lifespan. A deletion mutation in ser-1 significantly extended mean lifespan by up to 46% and maximum lifespan by 41% (Table 2). The life extension of the ser-1 mutant was suppressed by expression of the ser-1 plasmid (Table 2). While the mod-1 mutation had no effect on lifespan (Fig. 1a), a deletion mutation in ser-4 shortened early to mid lifespan (from 0 to 15 days after hatching; P = 0.01); the life-shortening effect of ser-4 was not significant during overall lifespan (P = 0.07). There was no clear abnormality in morphology observed in the ser-4 animals (data not shown). We suggest that serotonin receptors, ser-1 and ser-4, antagonistically affect lifespan.
The life-extension phenotype of the ser-1 mutant was largely suppressed by daf-16(RNAi) (Fig. 1e; Table 2). In addition, the ser-1 mutant did not increase extended lifespan of the daf-2(RNAi) animals. The life extension by ser-1(RNAi) was additive to that of the eat-2 mutant, which causes calorie restriction (Fig. 2a). It has been shown that there was no significant difference between the rate of pharyngeal pumping in the ser-1 animals and the wild-type animals, although there is a slight increase in variability in the rate (Hobson et al., 2006). Thus, the results suggest: (i) the ser-1 serotonin signal interlocks into the insulin/insulin-like growth factor 1 (IGF-1) pathway; (ii) the ser-1 life extension is independent of calorie restriction. It is yet to be determined how the serotonin pathway interacts with the insulin/IGF-1 pathway. Note that the long-lived ser-1(RNAi) animals did not show altered expression of an insulin-like peptide gene marker, daf-28::GFP (Supplementary Fig. 1). Similarly, ser-4(RNAi) did not affect expression of daf-28::GFP (data not shown). We also tested the effect of the mod-5 mutation that reduces removal of serotonin. Although the mod-5 mutation reduced the lifespan of the ser-1 mutation (Fig. 2b), it is likely that reduced lifespan of the mod-5 mutation causes this result.
Interestingly, the tph-1 mutant showed life extension by 20% in the presence of FUDR (Fig. 2c) but not under normal growth conditions (Fig. 1a). FUDR is known to suppress DNA synthesis, thus knocking out proliferating cells in reproductive tissues, eggs, and larvae. It is unlikely that internal hatching shortened lifespan in tph-1 under normal conditions, because we did not observe any contribution of such internal hatching to life-shortening. The life extension by the tph-1 mutation but not by the ser-1 mutation was suppressed by the supplementation of serotonin (P < 0.002; Fig. 2c,d; Table 2). The tph-1 and ser-1 mutations conferred resistance to heat and ultraviolet (UV) (Fig. 3).
In this study, we tested the hypothesis that serotonin signal influences lifespan based on observations in the fruit fly and in the mouse (De Luca et al., 2003; Mattson et al., 2004). Surprisingly, the major effects on lifespan were not observed in the mutants of the serotonin biosynthesis genes, but were observed in the mutants of the two serotonin receptors. The ser-1 and ser-4 mutants showed opposing effects on lifespan. This finding suggests a model in which altering levels of serotonin could trigger both life-extending and life-shortening effects depending on the serotonin receptors (Supplementary Fig. 2). In this model, reducing serotonin by the tph-1 mutations triggers the opposing effects on lifespan, suggesting that the tph-1 mutations do not necessarily cause the life-extension phenotype similar to ser-1 (ok345) and ser-1(RNAi). Note that the daf-16 nuclear localization is affected by the tph-1 mutant in response to starvation (Liang et al., 2006). This model also does not contradict the result that the mod-5 mutation caused a modest life-shortening effect. Interestingly, the ser-1 serotonin receptor gene is an example of the genes that have been missed in the genome-wide RNAi (RNA-mediated gene inactivation) screenings for lifespan (Dillin et al., 2002; Lee et al., 2003; Hamilton et al., 2005; Hansen et al., 2005). In fact, the rate of false negatives in an RNAi screening is relatively high (10–30%) even within the same laboratory (Simmer et al., 2003). This study suggests serotonin receptors as potential therapeutic targets for the prevention of age-related diseases and for anti-aging interventions.