Enhancing mask activity in dopaminergic neurons extends lifespan in flies

Abstract Dopaminergic neurons (DANs) are essential modulators for brain functions involving memory formation, reward processing, and decision‐making. Here I demonstrate a novel and important function of the DANs in regulating aging and longevity. Overexpressing the putative scaffolding protein Mask in two small groups of DANs in flies can significantly extend the lifespan in flies and sustain adult locomotor and fecundity at old ages. This Mask‐induced beneficial effect requires dopaminergic transmission but cannot be recapitulated by elevating dopamine production alone in the DANs. Independent activation of Gαs in the same two groups of DANs via the drug‐inducible DREADD system also extends fly lifespan, further indicating the connection of specific DANs to aging control. The Mask‐induced lifespan extension appears to depend on the function of Mask to regulate microtubule (MT) stability. A structure–function analysis demonstrated that the ankyrin repeats domain in the Mask protein is both necessary for regulating MT stability (when expressed in muscles and motor neurons) and sufficient to prolong longevity (when expressed in the two groups of DANs). Furthermore, DAN‐specific overexpression of Unc‐104 or knockdown of p150Glued, two independent interventions previously shown to impact MT dynamics, also extends lifespan in flies. Together, these data demonstrated a novel DANs‐dependent mechanism that, upon the tuning of their MT dynamics, modulates systemic aging and longevity in flies.


| INTRODUC TI ON
The complex process of aging has a reciprocal interaction with the functions of the brain. This connection was indicated by studies of the neuronal-specific genes and their ability to affect lifespan in animals (Pasyukova et al., 2015), the sensory functions of the nervous system in sensing and coordinating the responses to environmental cues that lead to impact on aging and longevity in animals (Alcedo & Kenyon, 2004;Apfeld & Kenyon, 1999;Bishop & Guarente, 2007;Chen et al., 2016;Gendron et al., 2014;Lee & Kenyon, 2009;Libert et al., 2007;Waterson et al., 2014;Zhang, Gong, et al., 2018), and the neuroendocrine system that modulates aging and longevity (Broughton et al., 2005(Broughton et al., , 2010Satoh & Imai, 2014;Zhang et al., 2013Zhang et al., , 2017. A recently emerging theme for the brain-driven regulation of aging highlights the change of longevity induced by neuronal cell-type-specific modulations. Recent studies showed Forkhead family transcription factors that are differentially expressed in the glia and neurons each enable distinct capacities to affect longevity (Bolukbasi et al., 2021). Specific neuronal types such as the serotonergic neurons in both worms (Higuchi-Sanabria et al., 2020;Zhang, Wu, et al., 2018) and flies (Chakraborty et al., 2019;Lyu et al., 2021;Ro et al., 2016) regulate aging through targeting different physiological functions in the peripheral tissues.
Dopaminergic neurons (DANs), a crucial modulatory system in the brain (Arias-Carrión et al., 2010;Schultz, 2007), have also been implicated in aging regulation. Limited studies on this topic yielded controversial results: On the one hand, the function of the dopaminergic system in regulating reward-feeding, food intake, and energy balance (Doan et al., 2016;Murray et al., 2014;Narayanan et al., 2010) implies an intuitive link to the control of aging. The results of a few previous studies support such a link: In rodents, increasing dopamine level leads to extended longevity (Cotzias et al., 1974;Knoll, 1997). In flies, polymorphism in dopa decarboxylase (Ddc), an enzyme required for dopamine biogenesis, correlates with variations in longevities in fly populations (De Luca et al., 2003). In worms, circumventing the ER unfolded protein stress in the DANs can moderately extend lifespan (Higuchi-Sanabria et al., 2020). On the other hand, loss of DANs or dopamine production in the brain does not seem to affect longevity. Depletion of dopamine production in the fly brains (Riemensperger et al., 2011) or in worms (Murakami & Murakami, 2007) does not affect lifespans in either organism. The life expectancy of human patients with Parkinson's disease (PD) who lose their DA neurons in the substantia nigra is similar to the normal population (Savica et al., 2017). Studies in humans and rodents also showed that chronically and globally increasing dopamine levels led to adverse effects (Stansley & Yamamoto, 2015). For example, long-term supplementation of L-DOPA together with deprenyl at low dosages causes an increased death rate in patients at an early stage of Parkinson's disease (Lees, 1995). Therefore, it remains elusive as to whether and how the dopamine system can actively affect aging.
My studies on a putative scaffolding protein, Mask, provided new evidence supporting a function of DANs in regulating aging and longevity. Mask was previously implicated in diverse signaling pathways and cellular processes. In mitotic cells, Mask acts as a modulator of the receptor tyrosine kinase signaling in flies , a co-factor of the Hippo pathway effector Yorkie (Sansores- Garcia et al., 2013;Sidor et al., 2013) and a positive regulator of the JAK/STAT pathway (Fisher et al., 2018). Our studies of Mask in the post-mitotic cells, such as muscles and neurons, demonstrated that it modulates mitochondrial morphology (Zhu et al., 2015), autophagy (Zhu et al., 2017), microtubule (MT) dynamics, and synaptic morphology (Martinez et al., 2021). In this study, I show that overexpressing Mask in two small groups of DANs in the fly brain significantly extends the lifespan of these flies, and this DAN-mediated effect can be independently recapitulated by moderately activating Gα s in the two groups of DANs. I also provide multiple pieces of evidence to support a model that enhanced MT dynamics in the DANs is a primary contributing factor conveying dopaminergic-dependent lifespan extension. All these results together point to the notion that MT stability and dynamics in specific DANs are targetable processes that can be tuned to impact aging.

| Small subsets of the DANs regulates longevity in flies
Our previous studies on Mask uncovered diverse functions of this putative scaffolding protein in the post-mitotic cells, including a strong protective effect against neurodegeneration induced by toxic protein aggregates (Zhu et al., 2017). In the continued investigation seeking to understand Mask's functions in neurons, I uncovered an interesting gain-of-function effect of Mask in the DANs.
Overexpressing Mask in DANs (driven by pan-dopaminergic Ddc-Gal4 or TH-Gal4 drivers) leads to an over 40% increase in lifespan in flies (median lifespan in male flies increased from ~76 days to ~104 days by Ddc-Gal4, and from ~72 days to ~105 days by TH-Gal4; Figures S1 and S2). Overexpressing Mask in either the entire body or the nervous system showed no effects on the lifespan ( Figure   S2), suggesting that other neuronal types potentially counteract the effects of DNAs on aging ( Figure S3). Dopaminergic neurons in the adult fly brain cluster into eight groups (Mao & Davis, 2009), and using three well-characterized Gal4 drivers that together show largely non-overlapping expression in most of the dopaminergic neurons in the fly brains (Burke et al., 2012;Galili et al., 2014;Liu et al., 2012), I tested whether all or only subsets of dopaminergic neurons are responsible for the Mask-mediated lifespan extension.
Overexpressing Mask in these three groups of dopaminergic neurons yielded distinct results on longevity. Overexpressing Mask in the PAM dopaminergic neurons marked by the 0273-Gal4 driver does not alter the lifespan ( Figure 1a); however, expressing Mask driven by either the TH-C'-or TH-D'-Gal4 drivers significantly extends the lifespan-the median lifespan in male flies increased from 64 to 96 days, and from 63 to 86 days, respectively (Figure 1b,c). Although the dopaminergic system provides essential modulation on various behaviors and physiological functions, flies devoid of dopamine in their brains (Riemensperger et al., 2011) and worms lacking the rate-limiting enzymes for dopamine synthesis both live a normal lifespan (Murakami & Murakami, 2007). Consistently, blocking neuronal activity in the TH-C' or TH-D' dopaminergic neurons by the DREADD-Di (Becnel et al., 2013) does not affect the lifespan in flies ( Figure S4). Together, these results suggest that dopamine systems may be dispensable for the mechanisms that drive the normal aging process. Overexpressing Mask in the specific dopaminergic neurons possibly induces a gain-of-function cellular effect, which consequently confers a beneficial outcome on aging and longevity.
Before investigating the underlying mechanisms induced by Mask, I first set out to determine whether activation of the DANs through approaches independent of Mask overexpression could also yield | 3 of 15 TIAN similar effects on longevity. I found that continuous full activation of the TH-C' or TH-D' dopaminergic neurons via the thermosensitive cation channel TrpA1 shortened the lifespan in the male flies and had no effect on the female flies ( Figure S5). However, moderate activation of these DANs induced lifespan extension in the flies ( Figure 2).
Feeding 1 μM Clozapine N-oxide (CNO; a concentration significantly below the full activation dose (Becnel et al., 2013)) to flies expressing this engineered Gα s receptor in their TH-C' or TH-D' neurons induces prominent and consistent lifespan extension ( Figure 2). These results further confirm the potentials of the DANs to impact lifespan.

| Dopamine transmission is required to elicit the Mask-induced lifespan extension
The Tyrosine Hydroxylase (TH)-positive neurons often use dopamine as the neural transmitter to communicate with other neurons but they may also co-transmit additional transmitter (Aguilar In order to achieve the upregulation, the flies were fed with dopamine precursor L-DOPA (Kayser et al., 2014), dopamine D1 receptor agonist SKF-82958 (Walters et al., 2002), dopamine D2 receptor agonist Quinpirole (Wiemerslage et al., 2013), or the D1 and D2 agonists together. It appeared that none of the treatments was able to extend the lifespan in flies. The lifespan of female flies fed with L-DOPA was even moderately shortened ( Figure S6). This adverse effect may be attributable to the global elevation of dopaminergic signaling, as long-term treatment with L-DOPA has been shown to cause neural toxicity in mammals (Stansley & Yamamoto, 2015). I next limited the intervention in specific DANs. Dopamine production was selectively increased in the TH-C' or TH-D' DANs via overexpressing Tyrosine Hydroxylase (TH) or Dopa Decarboxylase (Ddc) in these two groups 1 8 2 7 3 6 45 5 4 6 3 72 0 8 1 90 9 9 108 p < 0.001

| Tuning microtubule (MT) stability in the DANs induces lifespan extension in flies
In a parallel study, we discovered that Mask promotes microtubule (MT) dynamics in fly larval motor neurons and body wall muscles (Martinez et al., 2021). I next tested whether altered MT dynamics in the Mask-expressing DANs is the key mediator for lifespan extension. Mask is a large protein (4001 amino acids) with many func-   (Lazarus et al., 2013), are two independent interventions that have been previously shown to increase MT dynamics (Li et al., 2016), and overexpressing Unc-104 in the nervous system has also been shown to moderately extend lifespan in worms (Li et al., 2016). In flies, the lifespan was extended by ~37% when Unc-104 is overexpressed in the TH-C' DANs, and ~40% in the TH-D' DANs ( Figure 8a). Reducing p150 Glued levels in the DANs exhibited similar but milder effects only when it is applied to the TH-C' DANs-the lifespan was extended by ~14%. These results together further demonstrate that increasing MT dynamics in the TH-C' or TH-D' DANs is sufficient to induce lifespan extension in flies.

| DISCUSS ION
In this study, I demonstrated that overexpressing the putative scaffolding protein Mask in two small groups of the DANs has a profound effect on longevity. Mask overexpression likely causes a gain-offunction effect that impacts the state of MT stability in these DANs.
This effect would then induce a range of cellular changes that ultimately lead to altered neuronal outputs that can be relayed to the peripheral tissues, resulting in improved aging and prolonged lifespan. These findings uncovered a novel mechanism that modulates aging, but in order to fully understand this mechanism, many ques- is the control of reward-feeding and energy balance at the whole animal level (Doan et al., 2016;Murray et al., 2014;Narayanan et al., 2010). However, it is unclear whether DANs affect aging through the regulation of feeding and metabolism. Our analysis on the Mask-overexpressing long-lived flies indicated that the TH-C' and TH-D' DANs do not directly affect the amount of food consumption ( Figure S6). At the same time, the whole-body TAG levels in these flies are elevated ( Figure S7), instead of being depleted as was showed no consistent and relatable reduction of Dilp transcript levels in their brain ( Figures S9-S12). This result is somewhat consistent with the finding that disruptions of the insulin pathway in worms enhance lifespan but have no effect on the age-dependent decline of dopamine levels (Yin et al., 2014).
It is intriguing that the prolonged lifespan of the Maskoverexpressing flies is accompanied by a sustained fecundity in the long-lived flies ( Figure 6). Similar phenomena were also previously discovered by a number of other studies (Grandison et al., 2009;Hwangbo et al., 2004;Marden et al., 2003;Simon et al., 2003).
Moreover, the reproductive female of eusocial insects shows acquirable expansion of reproduction and lifespan. Together, these phenomena challenge the long-noticed theory about the tradeoff between reproduction and longevity, and suggest that a mechanism capable of extending both reproduction and longevity may exist as a common strategy for animals to actively intervene the aging process in order to cope with their reproductive demand. DANs, as critical modulators for brain functions, may be a vital part of a mechanism coordinating the sustainment and fulfillment of reproduction in animals. Future investigations that delineate the roles of DANs in regulating aging and reproduction can provide insight into the molecular and cellular basis for such a mechanism.
Mask and its mammalian homologs have been shown to play diverse functions in cells, including signal transduction (Fisher et al., 2018;Smith et al., 2002), transcription (Sansores-Garcia et al., 2013;Sidor et al., 2013), autophagy (Zhu et al., 2017), and vesiculation (Kitamata et al., 2019). Our most recent study demonstrated that Mask is also a potent regulator of MT stability (in press). Which of these functions contribute to Mask-induced lifespan extension? The

| Fly longevity assay
The procedures are adapted from the previously described protocol (Linford et al., 2013). 15-20 male or female flies were separated into one vial, and a total of 4-6 vials of flies were used for each genotype for longevity measurement. The flies are maintained at 25°C on standard food or food containing drugs and are transferred into fresh vials every 3 days.

| DREADD activation
10 mM Clozapine N-oxide (CNO; Tocris 4936) stock solution was prepared in DMSO. Food containing control (DMSO only) or 1 µM CNO was prepared freshly every three days before the flies were transferred to new vials during the longevity recording experiments.

| Fly fecundity assay
Newly eclosed individual virgin male or female flies (day1) were crossed with 2-3 w 1118 virgin female or male flies respectively and kept together for 3 days. The total progeny produced within the 3day intervals were counted after they eclose as adult flies. Sexually exposed and active male or female flies reared together with w 1118 female or male were aged till day 10, 30 and 50. Each female fly was then separated into a single vial and allow to lay eggs for 3 days.
The total progeny produced within the 3-day intervals were counted after they eclose. The aged male flies were individually mated with three 5-to 7-day-old w 1118 virgin females for 3 days, and fecundity was measured as the percentage of males that can successfully produce progenies.

| Statistical analysis
Data analysis for longevity: The Kaplan-Meier estimator was used to analyze the data and the survivorship curves were generated in Origin. The log-rank test was performed with Evan's A/B tools.
Each sample was compared with other samples in the group (more than two) using one-way ANOVA, or with the other sample in a group of two using a t test. The graphs were generated in Origin (Origin Lab). McGehee for editorial assistance. This work is supported by a startup fund from LSUHSC to X.T.

CO N FLI C T O F I NTE R E S T
The author certifies that she has NO affiliations with or involvement in any organization or entity with any financial interest, or nonfinancial interest in the subject matter or materials discussed in this manuscript.

AUTH O R CO NTR I B UTI O N S
X.T. is responsible for experimental design, data collection, and analysis. X.T. prepared the manuscript.

O PE N R E S E A RCH BA D G E S
This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. The data is available at https://www.biorx iv.org/ conte nt/10.1101/2020.06.15.153056v2.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available in BioRxiv at https://www.biorx iv.org/conte nt/10.1101/