Many molecular, cellular, and physiological processes in most organisms are coordinated with the predictable changes of the 24-h solar day. The circadian clock provides the mechanism of time keeping that is based on a negative feedback loop of transcriptional activators and repressors that generate endogenous molecular oscillations of circa 24 h (Hardin, 2011). A core repressor in the clock mechanism is encoded by the gene period (per) and the translocation of the PER protein into the cell nuclei followed by its degradation is the fundamental feature of clock function (Hardin, 2011). In both mammals and Drosophila, the circadian system consists of central and peripheral clocks (Hardin and Panda, 2013). Central pacemaker neurons in mammals are located in the suprachiasmatic nuclei (SCN). The central pacemaker neurons driving behavioral rest/activity rhythms consist of a network of about 150 neurons in the Drosophila brain. In addition to these central pacemaker neurons, mammals have intrinsic peripheral clocks in cells of fat tissues, kidneys, liver, and most other organs. Many of these peripheral tissues that express autonomous oscillators coordinate local tissue-specific processes. Similarly to mammals, peripheral clocks are widespread in fly tissues and function independently of the central pacemaker coordinating tissue-specific physiological processes (Giebultowicz, 2001). Within the nervous system, peripheral clocks are present in retinal photoreceptor cells and in other sensory neurons. In addition to neurons, some glial cells rhythmically express circadian clock genes in both mammals (Prolo et al., 2005; Marpegan et al., 2011; Hayashi et al., 2013) and in Drosophila (Ng et al., 2011). Early studies showed that PER protein is expressed in non-neuronal cells (Zerr et al., 1990; Ewer et al., 1992) and suggested that PER expression in these presumed glia is sufficient for manifestation of behavioral rhythmicity (Ewer et al., 1992). However, which glial cell subtypes express the PER-based oscillator and what their roles may be in the timekeeping processes remain poorly understood.

As organisms age, circadian rhythms tend to dampen as demonstrated in behavioral and molecular experiments both in mammals (Reddy and O'Neill, 2010) and Drosophila (Giebultowicz and Long, 2015). This phenomenon is implicated in declining cellular homeostasis and in various pathologies of aging, including altered inflammatory responses (Fonken et al., 2016), neurodegenerative diseases (Musiek et al., 2013) and impaired memory formation (Kondratova et al., 2010). In addition, physiological aging and late life diseases are accelerated by chronic disruption of clock functions in mammals (Kondratov et al., 2006; Antoch et al., 2008; Yu and Weaver, 2011; Hastings and Goedert, 2013). Similar to mammals, mutations in core clock genes accelerate aging phenotypes in Drosophila (Krishnan et al., 2009). Disruptions of the circadian clock in flies predispose them to neurodegeneration, although it is not known which clocks are involved (Krishnan et al., 2009, 2012). It was shown that Drosophila per⁰¹ mutants have increased levels of oxidative damage and neurodegeneration compared to age-matched controls (Krishnan et al., 2009). However, it is not known to what extent per mRNA is expressed in the glia, and consequently, whether loss of per in these cells could contribute to neurodegeneration and aging in general.

Glial cells play important roles in such processes as neuronal guidance during development, neuronal homeostasis, clearance of damaged tissues, and neurotransmitter recycling (Freeman and Doherty, 2006; Edwards and Meinertzhagen, 2010; Stork et al., 2012). Recent studies implicate mammalian astrocytes in neuroprotection via involvement in toxin clearance from the brain during sleep (Xie et al., 2013) and removal of damaged mitochondria from neurons in the process of transmitophagy (Davis et al., 2014). Glial cells were first classified based upon their location within the brain as surface, cortex, and neuropil glia. Recent classifications in mammals include astrocytes, microglia, oligodendrocytes, and Schwann cells. The Drosophila adult central nervous system (CNS) has five glial subtypes divided into three main categories, namely surface, cortex or neuropil glia.

Surface glia consist of two distinct subtypes, the perineurial and subperineurial glia. Perineurial glia are narrow, oblong cells that make up the outermost covering of the adult CNS (Awasaki et al., 2008). During development, these cells increase their cell division to maintain complete coverage of the adult Drosophila nervous system (Avet-Rochex et al., 2012). The function of this glial cell subtype in the adult fly brain remains largely unknown (Edwards and Meinertzhagen, 2010), but a recent study suggests that perineurial glia may be important in the transport of trehalose (the main energy supplying carbohydrate in insects) across the blood-brain barrier (Volkenhoff et al., 2015). Subperineurial glia are large, flat polyploidal cells (Unhavaithaya and Orr-Weaver, 2012) that reside just underneath the perineurial glia layer. Unlike perineurial glia, subperineurial glia undergo endoreplication during larval development to increase their cell size to maintain coverage of the brain through metamorphosis (Unhavaithaya and Orr-Weaver, 2012). These cells contain several tight septate junctions, form the blood-brain barrier of Drosophila, and separate the CNS from pathogens, xenobiotics, and the high electrolyte content of the hemolymph ultimately protecting neuronal function (Limmer et al., 2014; Weiler et al., 2017). Consistent with these functions, the transcriptome of surface glia is enriched for gene categories associated with drug metabolism, cell adhesion, and various transporters (DeSalvo et al., 2014).

Cortex glia make contact with the subperineurial glia through adherens junctions and envelope neuronal cell bodies that reside in the cortex providing metabolic support to them (Edwards and Meinertzhagen, 2010). One cortex glial cell can cover many neuronal bodies, which gives these cells a mesh-like appearance (Awasaki et al., 2008).

Located below the cortex are two types of neuropil glia, astrocytes and ensheathing glia. Ensheathing glia have a fibrous lamellar morphology (Awasaki et al., 2008) and act as phagocytes of the brain, similar to mammalian microglia. These glia respond to axonal injury through the Draper receptor signaling pathway (Doherty et al., 2009). Astrocyte glial cell bodies are located at the cortex/neuropil border and have projections that are closely associated with neuronal synapses and contain multiple neurotransmitter recycling pathways (Stork et al., 2012). A recent study of the transcriptome of fly astrocytes showed enriched expression of genes involved in metabolism, redox reactions, neurotransmitter synthesis and transport (Ng et al., 2016). RNAi-mediated knockdown of some of these genes revealed alterations in behavior including changes in activity level, activity onset, and mechanical stress induced paralysis (Ng et al., 2016).

It has been established that some glial subtypes express circadian clock genes in a rhythmic manner. In mammals, both astrocytes (Prolo et al., 2005; Marpegan et al., 2011) and microglia (Hayashi et al., 2013; Fonken et al., 2015) rhythmically express Per1 and Per2 proteins. Cultured astrocytes from Per1::luciferase transgenic rats and knock-in mice are capable of maintaining modest rhythms in circadian clock gene expression that can be entrained by physiologically relevant temperature changes (Prolo et al., 2005). Rhythmic expression of several clock genes was also shown in cortical microglia by qRT-PCR (Hayashi et al., 2013; Fonken et al., 2015). Expression of the circadian clock gene per in glia have been also suggested in flies (Ewer et al., 1992) and this was confirmed more recently although, it is not clear which glial subtypes express PER protein (Ng et al., 2011).

Although impairments of the circadian system are believed to be involved in accelerated aging, little is known about how the circadian clock in different tissues is altered across the lifespan. In Drosophila, PER expression remains robust in central pacemaker neurons (Luo et al., 2012) but is significantly reduced in retinal photoreceptors (Luo et al., 2012; Rakshit et al., 2012). While glia have many important roles in maintaining nervous system homeostasis, it is not known which glial subtypes express the core clock protein PER and whether PER levels remain similar across lifespan or decline with age. To address these questions, we took advantage of the fact that glial subtypes of Drosophila have unique expression patterns and can be labeled separately by GFP via cell-type specific drivers. We performed 2-timepoint immunocytochemical experiments to identify Drosophila glia subtypes that express PER protein and determined that the PER level in these cells is reduced in old fly brains compared to young.

We investigated which glia subtypes express the circadian clock protein PER in a manner similar to that of the pacemaker neurons and whether the relative amounts of PER signal change with age. To label specific glial cells, we employed the GAL4/UAS system (Brand and Perriman, 1993) using GAL4 lines to drive GFP expression in subtypes of glia with specific location and function in combination with immunocytochemistry to measure PER levels. It has been reported that PER expression in lateral and dorsal pacemaker neurons are equally strong in young and old Drosophila brains (Luo et al., 2012); therefore, the presence of PER staining in these neurons was used as a positive control. These cells have rhythmic PER expression with high levels at ZT22 and lack PER at ZT10 in wild type flies (Long et al., 2014); therefore, we selected these time points to examine glial cells.

In this study, we show that most glial cell subtypes of the adult Drosophila CNS express PER in a manner suggesting that circadian clock may function in these cells. These glial subtypes include perineurial glia, subperineurial glia, cortex glia, ensheathing glia of the central brain and the giant glia located in the medulla. While rhythmic PER expression in the medulla was reported previously (Suh and Jackson, 2007; Gorska-Andrzejak et al., 2009), our data suggest clock function in several other types of glial cells. However, the astrocyte glia appear to be an exception as we did not detect PER protein in these cells at either time point examined.

Our finding that perineurial and subperineurial glia express PER protein is consistent with a recent study of the surface glia transcriptome of adult Drosophila compared to the transcriptomes of all neurons, all glia, and to total brain lysates (DeSalvo et al., 2014). While not the focus of the study, their data do list per mRNA and other circadian clock genes as expressed in the surface glia (DeSalvo et al., 2014). Moreover, the core clock gene Clk was identified as one of the top 50 genes enriched in surface glia when compared to all glia (DeSalvo et al., 2014). The perineurial and subperineurial glia have distinct non-overlapping roles in the formation and maintenance of the blood-brain barrier that are not well understood (Awasaki et al., 2008; DeSalvo et al., 2014); the presence of the circadian clock in these cells may help to understand their functioning in the future.

Cortex glia constitute about 20% of the glia in the adult Drosophila brain (Kremer et al., 2017), but this subtype is relatively understudied in flies. Based on our results, cortex glia express PER protein similarly to the pacemaker neurons albeit at a much lower level even in young flies. However, we cannot exclude that cortex glia in other brain regions could show higher PER levels. Cortex glia are presumed to provide trophic support to the neuronal cell bodies they envelop (Edwards and Meinertzhagen, 2010). A recent study supports this idea by demonstrating that genes involved with β-oxidation are expressed in cortex glia suggesting that these cells may generate and transport ketone bodies (Schulz et al., 2015). Cortex glia are known to produce Ca⁺² oscillations and disruptions of these oscillations by mutations in the glial-specific Na⁺/Ca²⁺, K⁺ exchanger encoded by zydeco significantly decrease seizure threshold in flies (Melom and Littleton, 2013). While cortex glia are important for neuronal health and function, the role of potentially low amplitude PER oscillations in these cells has yet to be addressed.

Ensheathing glial cells are closely associated with neuronal arborizations and synaptic regions. We determined that these cells express PER in the central brain in Drosophila. It has been reported that the equivalent mammalian cells, the microglia express Per1 and Per2 in a circadian manner (Hayashi et al., 2013; Fonken et al., 2015). The ensheathing glia of Drosophila uniquely express key components of the glial phagocytic machinery such as the engulfment receptor Draper (Doherty et al., 2009). Interestingly, a recent study comparing circadian transcriptome in heads of young and old flies indicated that drpr mRNA show a rhythmic profile in young flies but the rhythm is dampened in old (Kuintzle et al., 2017).

We determined that the astrocyte glia in the central brain in the vicinity of the pacemaker neurons do not express PER. These results are consistent with a previous report that the astrocytes of the central brain are PER/TIM negative (Suh and Jackson, 2007). The absence of PER in fly astrocytes is somewhat unexpected given that mammalian astrocyte cultures from Per1::luciferase transgenic rats and knock-in mice are capable of maintaining modest rhythms in circadian clock genes expression (Prolo et al., 2005; Marpegan et al., 2011). In fact, several recent studies have demonstrated that mammalian astrocytes are important for controlling circadian timekeeping. Astrocyte-specific loss of core clock gene BMAL1 (the mammalian ortholog of cycle) via two independent methods was shown to alter circadian locomotor activity whereas expression of clock-associated kinase CK1ε in astrocytes was sufficient to lengthen the period of PER oscillations (Tso et al., 2017). Other recent studies also demonstrated astrocytes roles in circadian timekeeping through glia-neuron communication involving different signaling molecules (Barca-Mayo et al., 2017; Brancaccio et al., 2017). Consistent with the lack of PER in the fly central brain astrocytes, a study of the astrocyte transcriptome did not identify any of the core circadian clock genes (Ng et al., 2016). While astrocytes in both flies and mammals associate with neuronal synapses and have similar star-shaped morphologies and molecular markers, it is conceivable that circadian clock function could have been acquired later in evolution aided by the substantial proliferation of glia in mammals (Kremer et al., 2017).

Little is known about clock-controlled output processes in Drosophila glia. In the lamina of the fly visual system, glial cells show rhythmic changes in volume coordinated with the volume changes in the photoreceptor-contacting interneurons (Gorska-Andrzejak, 2013). These rhythmic changes in structure coincide with the rhythmic expression of the α-subunit of the sodium pump, Na⁺/K⁺-ATPase, which is in high abundance in glia and its rhythmic expression is per-dependent, as per⁰¹ mutants lack rhythmic expression of this subunit (Gorska-Andrzejak et al., 2009). A recent study implicates glial cell oscillators in the control of Gclc, a rhythmically expressed component of the rate-limiting enzyme in glutathione synthesis (Chow et al., 2016). This study reported that pan glial knockdown of the circadian clock gene cycle via loco-GAL4>cycRNAi was sufficient to significantly decrease rhythmicity of Gclc expression (Chow et al., 2016).

The ubiquitous and rhythmic expression of PER in glia reported here opens more questions regarding the functional significance of glial clocks. The role of glia in locomotor activity rhythms is not clear. Pan-glial knockdown of the circadian clock genes per or cryptochrome via RNAi failed to alter the free-running behavior of adult Drosophila (Ng et al., 2011). Yet, other studies have shown that Drosophila glial cell functions are required for behavioral rhythmicity (Jackson, 2011; Ng et al., 2011, 2016; Jackson et al., 2015). Finally, pan-glia knockdown of several astrocyte enriched genes can cause significant changes in activity level, sensitivity to mechanical stress, and/or alterations in circadian locomotor activity (Ng et al., 2016).

One of the important findings of our study is that, with the exception of the central brain neuropil glia, all PER expressing glial subtypes display significant age-related decline in PER protein levels. These data are consistent with the age-related decrease in PER protein reported by Western blot in whole heads (Luo et al., 2012; Rakshit et al., 2012; Kuintzle et al., 2017). Previous immunofluorescent studies detected PER decline in the retinal photoreceptors of old flies (Luo et al., 2012; Rakshit et al., 2012). Our data now show that similar decline occurs in the majority of glial cells. Although the reasons for age-related decrease in PER protein shown here are not known, it could be related to reported reduction of TIM protein in the heads of old flies (Luo et al., 2012; Rakshit et al., 2012). TIM protein is known to be required for PER stability (Hardin, 2011). It is also well known that PER is an essential repressor of CLK/CYC-activated circadian transcription of target genes (Hardin, 2011); therefore, our data suggest that the repressive arm of the circadian clock weakens in glia during aging due to decline of nuclear localized PER protein. Consistent with this hypothesis, a recent RNA-seq study showed that per mRNA expression is higher in the heads of old flies compared to young while protein levels are decreased in old (Kuintzle et al., 2017).

Our detailed analysis of PER expression suggests that circadian clock may function in several glial subtypes. These data should facilitate future functional analysis of glial circadian clocks and their roles in homeostasis of the nervous system. The age-related decline in PER protein expression in various glial subtypes may provide new ways of investigating the physiological processes that decline with age.

This study was conceived, analyzed, and written by DL and JG. All experiments were performed by DL.