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Published in final edited form as: Neurobiol Aging. 2019 Apr 11;80:83–90. doi: 10.1016/j.neurobiolaging.2019.04.004

Loss of DmGluRA exacerbates age-related sleep disruption and reduces lifespan

Sarah Ly 1, Nirinjini Naidoo 1,*
PMCID: PMC6679765  NIHMSID: NIHMS1529534  PMID: 31103635

Abstract

Declines in sleep amount and quality – characterized by excessive daytime sleepiness and an inability to sleep at night – are common features of aging. Sleep dysfunction is also associated with age-related ailments and diseases, suggesting that sleep is functionally relevant to the aging process. Metabotropic glutamate receptors (mGluRs) – which are critical regulators of neurotransmission and synaptic plasticity – have been implicated in both age-related disease and sleep regulation. Therefore, in this study, we examined the sleep and aging effect of complete genetic loss of mGluR signaling in Drosophila melanogaster. Genetic knockdown of the sole Drosophila mGluR - known as DmGluRA – reduced daytime wakefulness and nighttime sleep, recapitulating age-related sleep changes that occur across species. Furthermore, loss of DmGluRA significantly reduced lifespan and exacerbated age-related sleep loss in older flies. Thus, we identify DmGluRA as a novel regulator of sleep whose loss results in an age-relevant sleep phenotype that is associated with shortened lifespan. This is the first evidence that mGluR signaling regulates sleep/wake in a manner that is relevant to the aging process.

Keywords: metabotropic glutamate receptors, sleep, aging, lifespan, Drosophila

Introduction

Sleep is a critical component of health and wellness, regulating a broad range of physiological processes such as cognition (Dinges et al., 1997; Van Dongen et al., 2003), metabolism (Spiegel et al., 2009), and immunity (Prather et al., 2015). Declines in sleep quality and impaired ability to sleep are common features of aging (Reviewed in Cooke and Ancoli-Israel, 2011). Additionally, sleep dysfunctions are associated with higher risk for age-related dementia (Lim et al., 2013) (Pase et al., 2017) and neurodegeneration (Postuma et al., 2009) (Singletary and Naidoo, 2011) (Zhou et al., 2017). This suggests that sleep is functionally relevant to the aging process, though much remains unknown about what mechanisms may underlie such a relationship.

Metabotropic glutamate receptors (mGluRs) are g-protein coupled receptors that activate intracellular signaling cascades in order to modulate synaptic plasticity and neurotransmission (Conn and Pin, 1997). In mammalian systems, mGluRs exist as 8 different subtypes that can be classified into 3 groups (type I, II, or III) based on their amino acid sequence homology and signal transduction mechanisms (Niswender and Conn, 2010). There has been some evidence that dysregulation of various mGluR groups is associated with age-related decline. For example, reduced hippocampal expression of the mGluR2 subtype is correlated with memory impairments in aged rats (Ménard and Quirion, 2012). In Alzheimer’s disease subjects, both mGluR1 and mGluR2 downregulation has been observed in multiple disease-relevant brain regions (Albasanz et al., 2005; Richards et al., 2010), and the extent of mGluR1 downregulation in the cortex correlates with disease progression (Albasanz et al., 2005). These data suggest that mGluR signaling may be involved in aging processes in the brain. Furthermore, mGluRs have been implicated in sleep regulation from sleep studies in rodent animal models. Previous studies have examined the effects of subtype-specific knockdown of mGluRs and found that loss of different mGluR subtypes produces various changes in sleep and wake. Genetic loss of the group II mGluRs increases wakefulness and light-mediated shifts in circadian rhythms (Pritchett et al., 2015) while null mGluR5 mice exhibit altered sleep responses after sleep deprivation (Ahnaou et al., 2015). These results suggest that different mGluRs may regulate sleep though further analysis is required to exclude redundancy or compensatory effects from other mGluR subtypes that might occur following loss of any single mGluR.

Here, we sought to examine the role of mGluR signaling in sleep and aging in an animal model containing a single isoform of mGluR. We investigated the effects of complete genetic knockdown of mGluR in Drosophila melanogaster, also known at the common fruit fly. In contrast to mammals, Drosophila carries just one functional gene for mGluR, known as DmGluRA (Parmentier et al., 1996). The Drosophila animal model is an ideal system in which to study the relationship between sleep and aging, since Drosophila exhibits conserved features of sleep (Hendricks et al., 2000; Shaw et al., 2000) and also undergoes changes in sleep quality with age that are similar to those found in higher order species (Koh et al., 2006; Brown et al., 2014). We examined sleep behavior in flies that are null for the DmGluRA gene and found that loss of DmGluRA signaling in young flies reduces wakefulness during the day while reducing sleep at night, recapitulating the daytime sleepiness and nighttime insomnia effects of aged sleep (Reviewed in Cooke and Ancoli-Israel, 2011). Interestingly, while young null DmGluRA mutants do not exhibit any differences in total sleep in a 24-hour day, examination of sleep in aged flies reveals that null DmGluRA mutants become short sleepers later in life. Aged DmGluRA mutants display much greater age-related sleep loss than wildtype flies, and we also find that this is accompanied by a significantly shortened lifespan. Therefore, we identify DmGluRA as a novel regulator of sleep and demonstrate that loss of DmGluRA signaling has negative consequences on aging and lifespan.

Materials and Methods

Drosophila stocks and husbandry

Wildtype and mutant flies in the following study are in the white Canton-Special (wCS10) genetic background strain, which was originally a gift from Ronald Davis (Scripps Research Institute, Jupiter, FL). The DmGluRA null mutant carries the DmGluRA112 null allele (Bogdanik et al., 2004) and was a gift from Tom Jongens (University of Pennsylvania, Philadelphia, PA). The null DmGluRA mutant was outcrossed into the wCS10 laboratory background strain for 10 generations prior to all molecular and behavioral experimentation. All flies were raised on standard dextrose media (University of Pennsylvania Cell Center, Philadelphia, PA) and maintained in a 12 hour light:dark cycle at 25°C prior to and during all sleep recordings, except in those sleep recordings conducted in constant dark conditions (see Drosophila Sleep Assays). Female flies were utilized for all experiments to control for the effects of gender on behavioral outcomes.

Drosophila Sleep Assays

Flies were collected under CO2 anesthesia after eclosion and allowed to grow to one week of age before recording. For all sleep assays, female flies were placed in glass locomotor tubes containing standard dextrose media and allowed to acclimate for one full day in the recording chamber before the start of data collection. Sleep and wake were recorded by video and analyzed as previously described (Zimmerman et al., 2008). Sleep is defined as 5 or more minutes of continuous inactivity (Shaw et al., 2000). For constant dark condition recordings, flies were placed in a recording chamber in which all light had been removed and sleep was recorded for 7 days and nights starting on the following day.

Negative Geotaxis Assay

Negative geotaxis was measured as previously described (Ali et al., 2011). Briefly, negative geotaxis was observed in groups of 10 flies at a time during which flies were placed inside two conjoined plastic vials (Genesee Scientific, San Diego, CA) and gently tapped to the bottom of the lower vial. An 8cm demarcation was placed above the bottom of the plastic vial. For each trial, the number of flies passing the 8cm mark after being tapped to the bottom of the vial was recorded. Climbing rate was recorded in each group for a total of 10 trials with 1 minute of rest provided between trials.

Lifespan Assay

Female wCS10 wildtype and null DmGluRA female flies were collected under CO2 anesthesia for the lifespan assay. At one week of age, 140 flies were collected per genotype and separated into groups of 20 flies placed in separate vials containing standard dextrose media. Survival was scored every 3 to 4 days when flies were switched to fresh vials of standard dextrose media.

Western Blotting

Flies were sacrificed over dry ice. Pooled fly heads were homogenized in chilled standard lysis buffer (10mM Tris-HCl, 1mM EDTA, 10% Glycerol, 1% Triton-X, 150mM NaCl) containing Halt™ Protease Inhibitor Cocktail (ThermoFisher Scientific). Head lysates were centrifuged to remove cellular debris, and the concentration of proteins was measured with the Pierce micro-BCA assay (ThermoFisher Scientific). Protein was run on sodium dodecyl sulfate (SDS) polyacrylamide gels (10% Tris-HCl), transferred to nitrocellulose membranes (Bio-Rad), and incubated with DmGluRA 7G11 primary antibody (1:50, European Molecular Biology Laboratory, Heidelberg, Germany). Membranes were blocked with 5% milk and incubated with anti-mouse HRP secondary antibody (1:2500). Western blot analysis had been previously conducted in our lab to confirm the specificity of the DmGluRA antibody.

Statistical Analysis

Comparisons of sleep and wake outcomes were performed using T-tests (for two groups) or oneway analysis of variance (ANOVA; for >2 groups) followed by pairwise T-tests with Holm-Sidak correction for multiple comparisons if the global null hypothesis of no differences among groups was rejected (P<0.05). Unless otherwise noted, results are presented as mean and SEM. Categorical outcomes (e.g., distribution of bout lengths) were compared between groups using Chi-squared tests. In analyses of lifespan, survival curves were compared between genotypes using the log-rank test. When relevant, analyses of daytime and nighttime sleep were performed separately.

Results

Loss of DmGluRA reduces daytime wakefulness and nighttime sleep

Drosophila sleep has a diurnal rhythm consisting of sleep bouts that occur across both the day and night. Sleep bouts range in length from a few minutes to approximately a couple hours and are longest during the night (Hendricks et al., 2000). Sleep during the day – which occurs primarily in the middle of the day – is often referred to as a midday “siesta” (Wijnen and Young 2008). To assess the role of mGluR signaling in regulating sleep/wake behavior, we measured sleep/wake in Drosophila mutants lacking the Drosophila mGluR gene, DmGluRA. These mutants carry a null allele of DmGluRA and do not express DmGluRA protein (Bogdanik et al., 2004). After outcrossing the null DmGluRA mutant strain into the wildtype white Canton-Special (wCS10) genetic background, we reconfirmed that DmGluRA protein is not expressed in the null DmGluRA mutants (Figure 1A). We measured sleep and wake using a video tracking system that records activity in individual flies (Zimmerman et al., 2008). Periods of inactivity lasting 5 minutes or more are recorded as sleep, based on previous studies that demonstrate that this period of inactivity is associated with reduced arousal response (Hendricks et al., 2000) and brain activity in Drosophila (van Alphen et al., 2013). We found that null DmGluRA mutants have an altered daily profile relative to wildtype flies that is characterized by an increase in sleep during the day and a decrease in sleep during the night (Figure 1B and 1C). Genetic loss of DmGluRA did not change daily sleep amounts (Figure 1D). To confirm that null DmGluRA mutants are not simply hypoactive or hyperactive, we measured the rate of activity of the null DmGluRA mutants and confirmed that DmGluRA mutants did not exhibit any significant changes in activity compared to wildtype flies during the sleep experiments (Figure 1E). We also measured climbing ability of null DmGluRA mutants to confirm that the null mutants do not demonstrate any basal locomotor deficits compared to wildtype flies (Figure 1F).

Figure 1. Genetic loss of DmGluRA alters the distribution of sleep across the day and night.

Figure 1.

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(A) Null DmGluRA mutants do not express DmGluRA protein, as seen from the expression of the ~200kDa DmGluRA dimer that is expressed in wildtype flies and not in the null mutant. β-actin is shown and was used as a loading control (B) Daily sleep profile of wildtype wCS10 flies and null DmGluRA mutants over the course of 48 hours. Null DmGluRA mutants exhibit an altered distribution of sleep across the day and night compared to wildtype flies (N=70 flies per group, shaded area represents SEM) (C) Quantification of total sleep amount during the day and night. Sleep levels are higher during the day and lower during the night in null DmGluRA mutants relative to wildtype flies (N=70 flies per group, ***P<0.001) (D) Quantification of total sleep amount during the 24 hour day. Total amount of daily sleep is unchanged in the null DmGluRA mutant relative to wildtype flies (N=70 flies per group, NS = not significant, P=0.276 (E) Rate of activity is unchanged in null DmGluRA mutants relative to wildtype flies (N=70 flies per group, NS = not significant, P=0.2499) (F) Null DmGluRA mutants do not have impaired locomotor ability as measured by the climbing pass rate in a negative geotaxis assay (N=110 flies per group, NS = not significant, P=0.883) (G) Null DmGluRA mutants exhibit more sleep bouts during the day. Average daytime sleep bout length is not different between null mutants and wildtype flies. At night, sleep bout number and sleep bout length are not significantly different between null DmGluRA mutants and wildtype flies (N=70 flies per group, **P<0.01, ****P<0.0001) (H) Daytime wake bouts are increased in null DmGluRA mutants while average wake bout length is reduced. At night, wake bout number is not significantly changed in null DmGluRA mutants while average wake bout length is increased compared to wildtype flies (N=70 flies per group, **P<0.01, ****P<0.0001)

To further understand how sleep is altered in the null DmGluRA mutant, we examined sleep architecture in these flies by comparing the number and length of sleep and wake bouts to those parameters in wildtype flies. During the daytime, null mutants have a higher number of average sleep bouts than wildtype flies (Figure 1G) while the average length of sleep bouts is not significantly changed (Figure 1G). At night, despite the total reduction in nighttime sleep amount (Figure 1C) there was no statistically significant difference in the average number of sleep bouts or in the average sleep bout length between null DmGluRA mutants and wildtype flies (Figure 1G). We attribute this to a large variability in nighttime sleep bouts in both groups and upon further analysis of the distribution of average bout lengths in individual flies, we observe that a greater proportion of null DmGluRA mutants have an average sleep bout length that is less than 15 minutes during the night compared to wildtype flies (Supplemental Figure 1).

Analysis of wake architecture reveals that daytime wakefulness is fragmented in null mutants (Figure 1H). Null DmGluRA mutants have a larger number of wake bouts and the average wake bout length during the day is reduced in the mutant compared to wildtype flies. (Figure 1H). In contrast, the average length of nighttime wake bouts in the null mutant is significantly longer compared to wildtype flies (Figure 1H). This suggests that loss of DmGluRA causes an inability to maintain wake during the day (when wake is normally more consolidated) and sleep during the night (when sleep should be more consolidated).

DmGluRA mediates light-dependent allocation of daytime and nighttime sleep

Given the daytime- and nighttime-specific effect of changes in sleep following DmGluRA knockdown, we sought to determine how light entrainment might contribute to the DmGluRA mutant sleep phenotype. We therefore measured sleep in null DmGluRA mutants in the absence of external light cues. While null DmGluRA mutants initially demonstrate reduced wake during active periods and less sleep during inactive periods in the first few days in the dark, after multiple days without light cues the sleep and wake rhythms in null DmGluRA mutants become more similar to those in wildtype flies (Figure 2A). After multiple days without light, the sleep amount is similar during the subjective day and night between wildtype flies and null DmGluRA mutants (Figure 2B) and these changes are already observed after two days in constant dark (Supplemental Figure 2). This suggests that DmGluRA regulates behavioral state according to light onset and offset, maintaining wakefulness during light periods while promoting sleep during dark periods. Additionally, sleep and wake architecture is similar between wildtype flies and null DmGluRA mutants in constant darkness (Figure 2C and 2D), further demonstrating that the effects of DmGluRA knockdown on sleep and wake is dependent on light entrainment.

Figure 2. Null DmGluRA mutants have a similar sleep profile to wildtype flies in constant conditions.

Figure 2.

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(A) 7 day sleep profile of wildtype wCS10 flies and null DmGluRA mutants in constant darkness (N≥ 34 flies per group, shaded area represent SEM) (B) Quantification of total sleep (averaged over the last three days of recording) during the subjective day and night. Daytime and nighttime sleep amount is not significantly different between null DmGluRA mutants and wildtype flies in constant dark conditions (N≥ 34 flies per group, NS= not significant, Day: P=0.8063, Night: P=0.4607) (C) Null DmGluRA mutants do not exhibit any changes in the average number of sleep bouts during the subjective day and subjective night (averaged over the last three days of recording) compared to wildtype flies in constant dark conditions (N≥ 34 flies per group, NS= not significant, Day: P=0.1536, Night: P=0.5925) (D) Null DmGluRA mutants do not exhibit any changes in the average length of sleep bouts during the subjective day and subjective night (averaged over the last three days of recording) compared to wildtype flies in constant dark conditions (N≥ 34 flies per group, NS= not significant, Day: P=0.2364 Night: P=0.1373)

Loss of DmGluRA reduces lifespan

Since sleep/wake disruptions are known to negatively impact health, we sought to investigate the lifespan effects of DmGluRA knockdown in Drosophila. We conducted a survival assay of null DmGluRA mutants and wildtype flies and found that loss of DmGluRA is associated with a reduction in both median and maximum lifespan (Figure 3A). When comparing the survival curves between genotypes, null DmGluRA flies had significantly worse survival compared to wildtype flies (log-rank test P<0.0001). Average lifespan was reduced by more than 20% in null DmGluRA mutants compared to wildtype Drosophila (Figure 3B). Thus, in addition to altering normal sleep/wake patterns, loss of DmGluRA appears to impact the aging process of the fly.

Figure 3. Null DmGluRA mutants have reduced lifespan.

Figure 3.

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(A) Survival curves of wildtype flies and null DmGluRA mutants. Null DmGluRA mutants exhibited a shorter median lifespan compared to wildtype controls (Wildtype median age = 80 days; DmGluRA null median age = 66 days). Log-rank (Mantel-Cox) test was performed to determine statistical significance of difference between survival curves (N=140 flies per group, P<0.0001) (B) Average lifespan is significantly reduced in null DmGluRA mutants compared to wildtype flies (N=140 flies per group, error bars represent SEM, ****P<0.0001)

Loss of DmGluRA exacerbates age-related sleep loss

Following the observation that loss of DmGluRA reduces lifespan, we sought to determine if sleep in null DmGluRA mutants undergoes any age-specific changes that are different from wildtype flies. We examined sleep behavior in aged, one month old wildtype flies and null DmGluRA mutants since it has been found that during this time, Drosophila exhibit behavioral changes that are indicative of a shift to senescence (Carey et al., 2008). Here, we discovered that at one month of age, both wildtype and null DmGluRA mutants exhibit both daytime and nighttime sleep loss relative to young flies (Figure 4A). Notably, while young null DmGluRA mutants and wildtype flies exhibit no difference in daily sleep amount, aged null DmGluRA mutants sleep significantly less during the 24 hour day in comparison to wildtype flies (Figure 4B). This difference in sleep appears to be due to a dramatic reduction in nighttime sleep, since aged null DmGluRA mutants sleep more during during the day than wildtype flies (Figure 4C). Thus, aging is associated with both daytime and nighttime sleep loss as previously shown (Brown et al., 2014), but the loss of DmGluRA appears to exacerbate this effect, primarily due to a dramatic reduction in sleep during the night.

Figure 4. Age-related sleep loss is exacerbated in null DmGluRA mutants.

Figure 4.

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(A) Sleep profile of young wildtype flies at one week of age compared to aged wildtype and null DmGluRA mutants at one month of age (N≥15 flies per group, shaded area represents SEM) (B) Sleep per 24hr day. Both aged wildtype and aged DmGluRA mutants exhibit significant reductions in daily sleep amount compared to young wildtype flies. Aged DmGluRA mutants also exhibit significantly less daily sleep compared to aged wildtype flies. (N≥15 flies per group, ****p<0.0001) (C) Total daytime sleep and nighttime sleep are reduced with age. Both wildtype and null DmGluRA mutants display significant daytime and nighttime sleep loss at one month of age compared to one week old wildtype flies. At one month of age, null DmGluRA mutants sleep more during the day and less at night than one month old wildtype flies (N≥15 flies per group, * P<0.05, ****P<0.0001) (D) Daytime sleep bouts are reduced in both aged wildtype and aged null DmGluRA mutants compared to young wildtype flies (N≥15 flies per group, **P<0.01, ****P<0.0001). Average sleep bout length during the day is not significantly changed (N≥15 flies per group, P=0.4874) (E) Nighttime sleep bouts are not significantly different between young wildtype flies and aged null DmGluRA mutants, though they are reduced relative to aged wildtype flies, which exhibit an increase in nighttime sleep bout number relative to young wildtype flies. Nighttime sleep bout length is significantly reduced in aged null DmGluRA mutants relative to young wildtype (N≥15 flies per group, **P<0.01, ****P<0.0001) (F) With age, daytime wake becomes more consolidated in both wildtype flies and null DmGluRA mutants. Day time wake bouts are reduced while average wake bout length is increased in aged wildtype and null DmGluRA mutants compared to young wildtype flies (N≥15 flies per group, **P<0.01, ****P<0.0001) (G) At one month of age, both wildtype and null DmGluRA mutants have increased nighttime wake bouts and average wake bout length than young wildtype flies. Null DmGluRA mutants exhibit more frequent and longer wake bouts compared to aged wildtype flies (N≥15 flies per group, *P<0.05, ***P<0.001, ****P<0.0001)

Analysis of sleep architecture in aged flies revealed that with age, both wildtype and null mutants have fewer daytime sleep bouts compared to young wildtype flies with no significant change in average length of sleep bouts during the day (Figure 4D). In contrast, aged wildtype flies exhibit nighttime sleep fragmentation relative to young wildtype flies as measured by an increase in the number of sleep bouts and a decrease in average sleep bout length (Figure 4E) while aged DmGluRA null mutants exhibit decreases in the average sleep bout length at night (Figure 4E). Analysis of wake architecture in aged wildtype and DmGluRA null flies relative to young wildtype flies shows that wake is consolidated during the daytime (Figure 4F). At night, aged null DmGluRA mutants exhibit a significant increase in average wake bout length relative to both young and aged wildtype flies (Figure 4G).

Discussion

In this study, we demonstrate that loss of the Drosophila metabotropic glutamate receptor DmGluRA confers an aged-sleep phenotype and accelerates age-related sleep loss in the fly. Increased sleepiness during the day and an inability to sleep at night are common features of sleep in the elderly (Reviewed in Cooke and Ancoli-Israel, 2011) and it has been previously reported that aged Drosophila have sleep patterns that are less restricted to the night and more evenly dispersed across the 24 hour day/night cycle (Koh et al., 2008). Here, we demonstrate that genetic loss of DmGluRA leads to a redistribution of daily sleep; null DmGluRA mutants have higher amounts of daytime sleep and lower amounts of nighttime sleep compared to wildtype Drosophila. Thus, the sleep profile of young null DmGluRA mutants resembles an aged-sleep phenotype. The effect of DmGluRA on sleep in young flies does not persist in constant conditions, suggesting that DmGluRA regulates daytime wakefulness and nighttime somnolence in a light-dependent manner. Previous work has demonstrated that cell-specific genetic knockdown of DmGluRA in light-sensitive PDF clock cells in the Drosophila brain increases locomotor activity after light offset and before light onset (Hamasaka et al., 2007). Since we observed more wakefulness during these times in our experiments, it suggests that DmGluRA signaling from these clock cells are likely contributing to the observed sleep phenotype in the null mutant. Because DmGluRA protein is also broadly expressed throughout the Drosophila brain (Devaud et al., 2008), it is likely that mGluR signaling in other brain regions may also regulate sleep/wake, and future investigation will be necessary to address this.

An interesting finding in this study is that null DmGluRA mutants are short sleepers, but only with age. In young flies, loss of DmGluRA did not alter the total sleep over the 24 hour day. However, aged flies without DmGluRA sleep significantly less than aged wildtype flies. In this study, we found that aged wildtype and DmGluRA mutant flies displayed more consolidated wake than young flies during the daytime, in accordance with our prior observations previously reported in aged flies (Brown et al, 2014). While wake fragmentation rather than consolidation has been previously reported in aged animals, variations in our behavioral results compared with other literature may be due to our use of video analysis, which has been shown to be more accurate than Drosophila activity monitoring system (DAMS) measurements of sleep and wake, particularly during the day where wake may be underestimated by DAMS (Zimmerman et al. 2008). The overall significant decrease in sleep in the aged mutant flies compared to the aged wildtype flies suggests that loss of DmGluRA accelerates age-related sleep loss, rather than directly regulating sleep amount at all ages of development. Thus, it may not merely be sleep fragmentation or short sleep in early life that determines lifespan, but also the proper timing and distribution of sleep across the day that may be important for the aging process. The association between short sleep in aged null DmGluRA flies and a reduction in the average lifespan is supported by evidence across species. In humans, short sleep duration has been strongly associated with increased mortality (though it should also be noted that abnormally long sleep duration produces the same association) (Reviewed in Grandner et al., 2010) and a previous study on short-sleeping Drosophila mutants also found an association between higher wake amounts and increased mortality (Bushey et al., 2010). Certainly, what remains to be determined is whether sleep can be causally linked to aging outcomes and lifespan. For example, in the context of our current findings, it will be important to determine whether manipulations that increase sleep in aged null DmGluRA mutants could rescue the negative effects on lifespan.

In this study, aged flies were defined as flies that were one month old. Previous studies examining the aging effect on Drosophila sleep reported reduced sleep time and increased sleep fragmentation flies that were two months of age (Koh et al., 2006; Brown et al., 2014), or twice the age of the aged group in our study. For the purposes of this study, we chose to examine sleep in one month old flies in order to measure sleep as close to the onset of age-related changes as possible. At one month of age, behavioral senescence may be just beginning (Carey et al., 2008) and the rhythm strength of sleep has not diminished to the extent that is observed at two months of age (Koh et al., 2006). An analysis of sleep across the lifespan of Drosophila ananassae recently found 30-35 days to be the age when changes in sleep efficiency are first observed (Kaladchibachi et al., 2019), further supporting this notion. Since aging-induced reductions in locomotor behavior (Koh et al., 2006; Carey et al., 2008) may mask sleep differences between groups at more advanced ages, we believe that one month may represent a critical midlife time point where aged-related changes in Drosophila behavior should be observed in future studies.

How might metabotropic glutamate receptor signaling modulate the aging process? Like all cells in the body, neurons are vulnerable to the effects of age, as many homeostatic processes in the cell degrade with time (Nikoletopoulou and Tavernarakis, 2012) (Vayndorf et al., 2016). In young animals, glutamatergic receptor expression and calcium signaling in neurons changes across sleep and wake states (Lanté et al., 2011) (Bushey et al., 2015), which suggests that sleep may be important for regulating neural excitability and maintaining synaptic homeostasis. In addition to mGluRs, glutamate binds ionotropic receptors present on many different cell types (Meldrum, 2000). Ionotropic glutamate receptors such as AMPA receptors (AMPARs) and NMDA receptors (NMDARs) are transmembrane ligand-gated ion channels that mediate fast synaptic transmission (Traynelis et al., 2010). Analysis of mGluRs in cultured suprachiasmatic nucleus (SCN) neurons has demonstrated that mGluR activation inhibits ionotropic glutamate receptor-mediated calcium increases in the cell (Haak, 1999). Furthermore, mGluR activation leads to a stable reduction in the expression of AMPA receptors at the cell surface (Sanderson et al., 2011). Interestingly, a reduction in AMPA receptors at the membrane has been found to be a correlate of sleep (Lanté et al., 2011) and during sleep, mGluR signaling is necessary for memory consolidation (Diering et al., 2017). Thus, one interpretation for why loss of mGluR would exacerbate sleep loss is that without mGluR signaling, modulation of intracellular calcium and receptor trafficking is lost, leading to increased cellular excitability during periods when inhibitory modulation is required, such as during sleep.

The results of this study have important implications for our understanding of processes that are mediated by mGluR signaling. For example, mGluRs – including DmGluRA – are critical for learning and memory (Schoenfeld et al., 2013) (Diering et al., 2017). Thus, mGluR dysregulation may underlie both sleep and cognitive impairments in old age, which is supported by data that higher expression of mGluRs is associated with better cognitive outcomes in aged rodents (Ménard and Quirion, 2012). In the periphery, Drosophila DmGluRA signaling is required for development of the neuromuscular junction (NMJ). Null DmGluRA mutants have been previously shown to exhibit altered NMJ morphology and changes in cellular excitability at the NMJ (Bogdanik et al., 2004). A limitation of the experiments described in this study is that examination of sleep in a genetic null does not allow us to rule out changes in development or in peripheral signaling as contributors to the observed sleep phenotype. While we did not identify any changes in baseline locomotor ability or activity that would indicate a behavioral contribution of peripheral mGluR signaling in null flies, we cannot definitively attribute the sleep effects to brain-specific mGluR signaling. Additionally, we must also consider whether changes in development of glutamatergic synapses as a result of DmGluRA knockdown might mediate sleep behavior. Future directions will be to examine the effects of conditional genetic knockdown of mGluR signaling in the brain. Furthermore, as previously discussed, mammalian systems express multiple mGluR subtypes, and different mGluR subtypes may have distinct roles in regulating sleep and aging. Future investigation will be necessary to address this.

Identifying molecular regulators of sleep regulation may have important clinical implications for the treatment of age-related disease. There is a great deal of evidence linking poor sleep to negative health outcomes and better sleep quality to increased longevity and improved health in the elderly (Reviewed in Grandner et al., 2010). If sleep has functional consequences for aging, it is possible that therapies that improve sleep might concurrently improve other outcomes for age-related diseases such as Alzheimer’s or Parkinson’s disease, where sleep disturbances are common symptoms (Musiek et al., 2015) (Knie et al., 2011). Furthermore, addressing sleep dysfunction in early or mid-life might even prevent later decline. Metabotropic glutamate receptors have long been considered for their therapeutic potential (Reviewed in Vaidya et al., 2013), and thus may represent an avenue for sleep therapy development in the future.

Supplementary Material

1

Supplemental Figure 1. Frequency distribution of wildtype and null DmGluRA flies with short (less than 15 minutes) versus long (greater than 15 minutes) average sleep bout lengths (N=70 flies per group). The distribution of flies exhibiting short or long average sleep bout length during the day is not affected by genotype (N=70 flies per group, P= 0.2699). In contrast, at night, there is a statistically significantly effect of genotype on the distribution of flies with short sleep bout lengths of less than 15 minutes, with a greater number of null DmGluRA mutants exhibiting short sleep bout lengths at night compared to wildtype flies (N=70 flies per group, P<0.001).

2

Supplemental Figure 2. Differences in the sleep profile between wildtype Drosophila and null DmGluRA mutants become less pronounced after 2 days in constant darkness (A) Total sleep (averaged over the first two days of constant dark recording) during the subjective day is not significantly different between null DmGluRA mutants and wildtype flies. Sleep is reduced in null DmGluRA mutants compared to wildtype flies during the subjective night (N≥ 34 flies per group, NS= not significant, P=0.1515; **P<0.01) (B) Null DmGluRA mutants do not exhibit significant changes in the average number of daytime and nighttime sleep bouts during the subjective day and night (averaged over the first two days of constant dark recording) compared to wildtype flies in constant dark conditions (N≥ 34 flies per group, NS= not significant, Day:P=0.400, Night:P= 0.0459) (C) Null DmGluRA mutants do not exhibit any changes in the average wake bout length during the subjective day and night (averaged over the first two days of constant dark recording) compared to wildtype flies in constant dark conditions (N≥ 34 flies per group, NS= not significant, Day: P=0.1368, Night: P=0.2765).

Highlights.

  1. DmGluRA is required for the normal distribution of sleep and wake across the day and night

  2. Genetic loss of DmGluRA reduces lifespan

  3. Genetic loss of DmGluRA exacerbates age-related sleep loss

Acknowledgments

This project was supported by funding from NHLBI T32 HL07953 and NIA AG17628. The authors would like to thank Brendan Keenan for reviewing the statistical analyses in this manuscript.

Funding: AG17628; T32 HL07953

Footnotes

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Disclosure statement

The authors state that there are no actual or potential conflicts of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Supplemental Figure 1. Frequency distribution of wildtype and null DmGluRA flies with short (less than 15 minutes) versus long (greater than 15 minutes) average sleep bout lengths (N=70 flies per group). The distribution of flies exhibiting short or long average sleep bout length during the day is not affected by genotype (N=70 flies per group, P= 0.2699). In contrast, at night, there is a statistically significantly effect of genotype on the distribution of flies with short sleep bout lengths of less than 15 minutes, with a greater number of null DmGluRA mutants exhibiting short sleep bout lengths at night compared to wildtype flies (N=70 flies per group, P<0.001).

NIHMS1529534-supplement-1.jpg (1,019.5KB, jpg)
2

Supplemental Figure 2. Differences in the sleep profile between wildtype Drosophila and null DmGluRA mutants become less pronounced after 2 days in constant darkness (A) Total sleep (averaged over the first two days of constant dark recording) during the subjective day is not significantly different between null DmGluRA mutants and wildtype flies. Sleep is reduced in null DmGluRA mutants compared to wildtype flies during the subjective night (N≥ 34 flies per group, NS= not significant, P=0.1515; **P<0.01) (B) Null DmGluRA mutants do not exhibit significant changes in the average number of daytime and nighttime sleep bouts during the subjective day and night (averaged over the first two days of constant dark recording) compared to wildtype flies in constant dark conditions (N≥ 34 flies per group, NS= not significant, Day:P=0.400, Night:P= 0.0459) (C) Null DmGluRA mutants do not exhibit any changes in the average wake bout length during the subjective day and night (averaged over the first two days of constant dark recording) compared to wildtype flies in constant dark conditions (N≥ 34 flies per group, NS= not significant, Day: P=0.1368, Night: P=0.2765).

NIHMS1529534-supplement-2.jpg (1.4MB, jpg)

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