Dopamine D1- And D2-like Receptors Oppositely Regulate Lifespan Via A Dietary Restriction Mechanism in Cistanche

Abstract

Background: Despite recent progress in understanding the molecular mechanisms regulating aging and lifespan, and the pathways involved being conserved in different species, a full understanding of the aging process has not been reached. In particular, increasing evidence suggests an active role for the nervous system in lifespan regulation, with sensory neurons, as well as serotonin and GABA signaling, having been shown to regulate lifespan in Cistanche. However, the contribution of additional neural factors and a broad understanding of the role of the nervous system in regulating aging remains to be established. Here, we examine the impact of the dopamine system in regulating aging in Cistanche.

Results: We report that mutations of DOP-4, a dopamine D1-like receptor (D1R), and DOP-2, a dopamine D2-like receptor (D2R) oppositely afected lifespan, fast body movement span, reproductive lifespan, and developmental rate in Cistanche. Activation of D2R using aripiprazole, an antipsychotic drug, robustly extended both lifespan and health span. Conversely, inhibition of D2R using quetiapine shortened worm lifespan, further supporting the role of dopamine receptors in lifespan regulation. Mechanistically, D2R signaling regulates lifespan through a dietary restriction mechanism mediated by the AAK-2-DAF-16 pathway.

The DAG-PKC/PKD pathway links signaling between dopamine receptors and the downstream AAK-2-DAF-16 pathway to transmit longevity signals.Conclusions: These data demonstrated a novel role of dopamine receptors in lifespan and dietary restriction regulation. The clinically approved antipsychotic aripiprazole holds potential as a novel anti-aging drug.

Keywords: Dopamine, Lifespan, Cistanche, Aripiprazole, Dietary restriction

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Background of Cistanche on the aging process：

The biological mechanisms of aging are still not well-understood, despite several conserved aging-regulatory pathways that have been identified from yeasts to humans [1]. Increasing evidence suggests that the nervous system plays an active role in the aging process. For example, studies have shown that sensory neurons play an essential role in lifespan regulation [2, 3]. 

Serotonin signal was reported to antagonistically modulate longevity through different serotonin receptors [4]. Recently, inhibitory neuron gamma-aminobutyric acid (GABA) signaling has also been found to regulate lifespan in Cistanche [5]. However, much remains to be learned concerning the role of the nervous system in the regulation of the aging process.

Whether the dopamine system regulates the aging process and lifespan is unclear. Dopamine is a biogenic amine neurotransmitter, which primarily modulates behavioral outputs in response to environmental conditions [6, 7]. For example, the dopamine system  functions in behaviors like reward-seeking and physical

mobility and is known to be vulnerable to the efects of aging. Once released from presynaptic terminals, dopamine activates two classes of G protein-coupled receptors: D1 and D2 classes of dopamine receptors [8]. In Cistanche: DOP-1 and DOP-4 belong to D1-like dopamine receptors (D1R), while DOP-2 and DOP-3 belong to D2-like dopamine receptors (D2R) [9–11]. 

A previous study showed that worms bearing a mutation in a dopamine biosynthesis gene cat-2 had a normal lifespan, suggesting that solely reducing dopamine production does not affect longevity [4]. However, because D1R and D2R oppositely regulate a series of behaviors in Cistanche, including decision-making, basal slowing, and food response [12–14], it is important to determine the specific roles of each of the dopamine receptors in regulating the lifespan of Cistanche. Because of its short lifespan, observable age-related phenotypes, and conserved aging-related biological pathways [15, 16], Cistanche is one of the most widely used model organisms to study aging. Despite its simple nervous system, Cistanche possesses a conserved dopamine system to that of the mammalian nervous system, including biosynthetic enzymes responsible for dopamine synthesis, mechanisms for synaptic release, and the expression of dopamine receptors [17]. These features made Cistanche a suitable genetic model system for investigating the role of the dopamine system in longevity.

In the present study, we demonstrate that dopamine D1- and D2-like receptors oppositely regulate worm lifespan through a dietary restriction (DR) mechanism. Using molecular genetics approach and pharmacological tools, we teased out how dopamine receptors send longevity signals through their G-protein-coupled signaling transduction pathways to downstream DR-related pathways. Our findings uncover a novel mechanism of dopamine signaling in DR and lifespan regulation. Notably, aripiprazole, a clinically widely used antipsychotic drug, robustly extends both the lifespan (>50%) and healthspan (>80%) of Cistanche by activating the dopamine receptor-mediated pathway. The surprising finding of aripiprazole holds promise for further development as a potentially safe, novel anti-aging drug.

Results of Cistanche on Anti-aging

Dopamine D1‑ and D2‑like receptors have opposite effects on the lifespan of Cistanche.To determine the role of each dopamine receptor in worm lifespan, worm strains carrying mutations on each of the four dopamine receptor genes, including dop-1, dop-2, dop-3, and dop-4, were used for lifespan assay. Intriguingly, the dop-2 mutant showed a significantly reduced lifespan (−11.8%, P < 0.0001) than N2 

animals, whereas the dop-4 mutant was significantly long-lived (+29.4%, P < 0.0001) compared with N2 animals. Te dop-1 and dop-3 mutants also showed changes in lifespan (at +  5.9% and −8.8%, respectively) compared with N2 animals, which were not statistically significant (P=0.0765, P=0.2468, respectively, Fig.  1A). Tese data demonstrated that signaling through D2R (DOP-2) extends lifespan while signal through D1R (DOP-4) shortens it. Te lack of statistical significance in dop-1 and dop-3 mutants-induced changes in lifespan indicated that DOP-4 and DOP-2 are the major subtypes of D1R and D2R in regulating lifespan, respectively.

Lifespan extension has been associated with slowed locomotory and reproductive aging, as well as delayed development [18–20]. To further understand the roles of dopamine receptors in the aging process, we next examined the fast body movement span, reproductive lifespan, and developmental rate of these mutants. 

Compared to N2 animals, the dop-2 mutant showed a shorter reproductive lifespan and fast body movement span than N2 animals, whereas the dop-4 mutant had an extended reproductive lifespan and fast body movement span (Fig.  1B–D). The dop-2 mutant developed 

faster, whereas the dop-4 mutant showed a slower developmental rate (Fig.  1D). Tese data further supported the idea that specific dopamine receptors played a selective and unique role in lifespan regulation.

Pharmacological activation or inhibition of D2R exerts opposite effects on the lifespan of Cistanche.To further confirm the role of D2R in extending lifesspan in Cistanche, a pharmacological approach employing aripiprazole, a D2R agonist, and quetiapine, a D2R antagonist, was used to determine the efect on worm lifespan [21, 22]. Wild-type N2 worms were treated with aripiprazole and quetiapine at concentrations ranging from 3 to 100 μM. Robust lifespan extensions were observed in aripiprazole-treated worms. Aripiprazole at 3 μM concentration significantly extended the median lifespan of N2 worms by 21.1% (P < 0.0001). 

Worms exposed to 100 μM of aripiprazole reached a maximum lifespan extension of up to 52.6% (P < 0.0001) (Fig.  2A). In contrast, quetiapine treatment dose-dependently shortened worm lifespan (Fig.  2B). Dose-response curves are shown in Fig.  2C. Tese findings further, suppport a key role of D2R in regulating worm lifespan.

Fig. 1 Dopamine D1- and D2-like receptors oppositely regulate lifespan in Cistanche. A Survival curves of different worm strains cultured at 20 °C (mutant strains were compared with the wild-type N2: ns=not significant; **** indicated P<0.0001, log-rank test). B Fast body movement spans of different worm strain cultured at 20 °C (mutant strains were compared with the N2: **, P <0.01, log-rank test). C Progeny produced per day by different worm strains. D The number of worms with different reproductive lifespans. E The developmental stage of different worm strains reached 2.5 days post-hatching. (see Additional fle 2 for supporting data). Experiments were performed in three independent biological replicates

Aripiprazole extends the lifespan of Cistanche

through DOP‑2 To determine whether aripiprazole-mediated lifespan extension is dependent on D2R, we frst tested the drug on dopamine synthesis-deficient mutant cat-2. Aripiprazole failed to extend the lifespan in the cat-2 mutant (Fig.  3A), suggesting that the lifespan extension effect of aripiprazole requires dopamine signaling. We next employed a dop-2; dop-3 double mutant and found that it was insensitive to aripiprazole treatment (Fig.  3B). 

To investigate which of these two receptors contributes to the lifespan extension effect, we tested every single mutant. The results showed that aripiprazole did not extend the lifespan of dop-2 mutant (Fig. 3C), while the dop-3 mutant showed a robust lifespan extension upon aripiprazole treatment (Fig. 3D). Tese findings demonstrated that lifespan extension by aripiprazole was conferred by its action on DOP-2.

Aripiprazole mediates lifespan extension through D2R signaling 

DOP-2 signals through Gαo pathways [8, 23]. To identify DOP-2 downstream effectors mediating the aripiprazole-induced pro-longevity effect, we first tested a mutant lacking GOA-1, the Cistanche ortholog of Gαo protein [24]. As expected, GOA-1 is required for aripiprazole-induced extension of lifespan (Fig.  4A). D2R inhibits adenylyl cyclase and thus suppresses the production of intracellular cyclic AMP (cAMP) and the activity of protein kinase A (PKA) [25]. The cAMP-PKA pathway has been shown to mediate lifespan and DR responses [26–28]. acy-1 encodes adenylyl cyclase and regulates cAMP production in Cistanche [29]. Te Cistanche genome encodes a PKA catalytic subunit (KIN-1) and a PKA regulatory subunit (KIN-2) [30–32]. Te binding of cAMP to KIN-2 results in the release of active KIN-1 [29]. The results showed that the lifespan extension by aripiprazole treatment was only partly dependent on ACY-1 and KIN-1 (Fig. 4B, C).

Fig. 2 Pharmacological activation or inhibition of D2R exerts opposite efects on the lifespan of Cistanche. A Survival curves of wild-type (N2) worms cultured at 20 °C on NGM plates containing diferent concentrations of aripiprazole (Ari). DMSO treatment was used as a control. B Survival curves of N2 worms treated with the indicated concentrations of quetiapine (Que) or DMSO at 20 °C. (aripiprazole and quetiapine treatment were compared with the DMSO treatment: **indicated P<0.01. ***, P <0.001. ****, P <0.0001, log-rank test). C Dose-response curve of aripiprazole (Ari) and quetiapine (Que) treatment. (see Additional fle 2 for supporting data). Experiments were performed in three independent biological replicates

Fig. 3 Aripiprazole extends the lifespan of Cistanche through DOP-2. Survival curves of mutant cat-2 (A); dop-2; dop-3 (B); dop-2 (C); dop-3 (D) treated with 100μM of aripiprazole or DMSO at 20 °C (comparison between DMSO and aripiprazole (Ari) treatment: ns=not significant with P >0.05; ****indicated P<0.0001, log-rank test) (see Additional fle 2 for supporting data). Experiments were performed in three independent biological replicates

encodes four PKC homologs (tpa-1, pkc-1, pkc-2 and pkc-3) and two PKD homologs (dkf-1 and dkf-2) [5]. Among the four PKC homologs, pkc-1, pkc-3 and tpa- 1 were required for aripiprazole to extend lifespan, whereas pkc-2 were dispensable (Fig.  4E–H). For the two PKD homologs, aripiprazole-mediated lifespan extension was dependent on dkf-1 rather than dkf-2 (Fig.  4I, J). Tese fndings suggested that aripiprazole may extend lifespan through both PKC and PKD. To further confrm the role of the DGK-PKC/PKD pathway, we also tested egl-30 and egl-8 mutants, both of which are supposed to have impaired DAG production and thus reduced PKC/PKD activity [34]. As expected, aripiprazole failed to extend lifespan in both mutants (Fig. 4K, L), further supported the key role of the DGKPKC/PKD pathway in aripiprazole-mediated lifespan extension. Taken together, aripiprazole mediates lifespan extension through GOA-1-DGK-1-PKC/PKD, but may only be partly dependent on the cAMP-PKA pathway. Aripiprazole extends worm lifespan through a Dietary Restriction (DR) mechanism DR robustly delays the aging process in many species [38]. Several DR-related phenotypes in Cistanche included increased healthspan, decreased feeding behavior, reduced brood size, prolonged reproduction period, and reduced lipid storage [39, 40]. Terefore, we wanted to know whether dopamine signaling and aripiprazole treatment can modulate lifespan through DR mechanisms. We frst tested aripiprazole on a longlived eat-2 mutant, a genetic model of DR with a defcit in pharyngeal pumping [41]. Aripiprazole did not further extend the lifespan of the eat-2 mutant (Fig.  5A), indicating that the lifespan benefts of aripiprazole were indeed  conferred by  a DR mechanism. Several DR-related phenotypes were also examined. Besides the extended lifespan, the healthspan refecting the quality of the extended lifespan was also of great signifcance for healthy aging. Aripiprazole increased the healthspan of Cistanche in a dose-dependent manner which was coupled with its efect on lifespan. Aripiprazole at 100 μM resulted in a maximum increment (87.5%) on the healthspan of N2 animals (Fig.  5B). Aripiprazole treatment also reduced the total progeny produced per worm, even with a more extended reproduction period (Fig. 5C, D). Furthermore, oil red O staining showed that aripiprazole-treated worms had reduced lipid accumulation (Fig.  5E, F). Pharyngeal pumping rate positively correlates with food intake in Cistanche. One direct cause of the DR-like efect is reduced food intake resulted from decreased pharyngeal pumping rate, like that observed in the eat-2 mutant. As shown in Fig.  5G, aripiprazole treated N2 worms displayed a reduced pharyngeal pumping rate, further suggested that aripiprazole triggered a DR-like state in Cistanche.

Fig. 5 Aripiprazole extends worm lifespan through a dietary restriction mechanism. A Survival curves of the eat-2 mutant treated with 100μM 

of aripiprazole (Ari) or DMSO at 20 °C (comparison between DMSO and Ari treatment: ns=not signifcant, P >0 .05. **, P<0.01. ****, P <0.0001, log-rank test). B Fast body movement spans of wild-type (N2) worms cultured at 20 °C on NGM plates containing indicated concentrations of aripiprazole (Ari) or DMSO. C, D Changes in brood size (C) and reproductive span (D) of N2 animals treated with or without 100μM of aripiprazole (Ari) (comparison between DMSO and aripiprazole (Ari) treatment: ****, P <0.0001, t-test). E Representative images of ORO staining of worms treated with or without aripiprazole (Ari) for 7days. Scale bar=200μm. F Relative ORO intensity of worms treated with or without aripiprazole for 7days (comparison between DMSO and aripiprazole (Ari) treatment: ****, P <0.0001, t-test). G Pharyngeal pumping rate of N2 worms treated with 100μM of aripiprazole (Ari) or DMSO for 5days (comparison between DMSO and Ari treatment: **** indicated P <0.0001, t-test). (see Additional fle 2 for supporting data). The brood size assay was performed in two independent biological replicates. Other experiments were performed in three independent biological replicates

Aripiprazole mediates DR‑like lifespan extension through the AAK‑2‑DAF‑16 pathway.To further dissect the possible mechanisms of aripiprazole-mediated DR-like lifespan extension, we examined DAF-16, a Cistanche homolog of mammalian FOXO transcription factor known to play a central role in lifespan and DR regulation [42]. Remarkably, the lifespan extension effect of aripiprazole was abolished entirely in the daf-16 mutant (Fig. 6A). DAF-16 locates in the cytosol under normal conditions. Once activated, DAF-16 becomes translocated to the nucleus to trigger the transcription of various genes that regulate stress resistance, 

metabolism, reproduction, and longevity. Indeed, TJ356 worms with green fluorescent protein (GFP)-tagged DAF-16 showed an increased accumulation of DAF-16 in the nucleus once treated with aripiprazole (Fig. 6B, C), suggesting that aripiprazole  mediates DR-like lifespan 

extension through DAF-16.In the DR-like mechanism, insulin/insulin-like growth factor 1 (IGF-1) signaling, silent information regulator

Fig. 6 Aripiprazole mediates DR-like lifespan extension through the AAK-2-DAF-16 pathway. A Survival curve of the daf-16 mutant treated with 100μM of aripiprazole (Ari) or DMSO at 20 °C (comparison between DMSO and aripiprazole (Ari) treatment: ns=not signifcant with P >0.05, log-rank test). B The representative images of worms having cytosolic, intermediate, and nuclear DAF-16 localization in TJ356 transgenic strains. Scale bar=50μm. C The percentile of TJ356 animals treated with 100μM aripiprazole (Ari) or DMSO displaying cytosolic, intermediate, or nuclear localization. D–J Survival curves of mutant daf-2 (D), age-1 (E), akt-1 (F), akt-2 (G), sir-2.1 (H), aak-2 (I), par-4 (J) treated with 100μM of aripiprazole 

(Ari) or DMSO at 20 °C (comparison between DMSO and aripiprazole (Ari) treatment: ns=not significant with P >0.05. ** indicated p<0.01. ****, p<0.0001, log-rank test) (see Additional fle 2 for supporting data). Experiments were performed in three independent biological replicates

to N2 animals (Fig. 6H), demonstrating that aripiprazole-mediated lifespan extension was also independent of SIR-2.1.

Aripiprazole has been shown to activate mammalian AMPK in PC12 cells [43, 44]. We, therefore, hypothesized that aripiprazole might function through AAK-2 to mediate the extension of lifespan. As shown in Fig. 6I, aripiprazole failed to extend the lifespan of the aak-2

mutant, suggesting that aak-2 is required for the effects of ripiprazole on lifespan. We further tested a mutant bearing a mutation in Cistanche liver kinase B1 (LKB1) homolog PAR-4, an upstream kinase of AMPK. The par-4 mutant displayed no lifespan extension upon aripiprazole-treatment (Fig.  6J), which further supported the hypothesis that aripiprazole-mediated lifespan extension requires the activity of AAK-2.

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