FTD-associated mutations in Tau result in a combination of dominant and recessive phenotypes.

Although mutations in the microtubules-associated protein Tau have long been connected with several neurodegenerative diseases, the underlying molecular mechanisms causing these tauopathies are still not fully understood. Studies in various models suggested that dominant gain-of-function effects underlie the pathogenicity of these mutants; however, there is also evidence that the loss of normal physiological functions of Tau plays a role in tauopathies. Previous studies on Tau in Drosophila involved expressing the human Tau protein in the background of the endogenous Tau gene in addition to inducing high expression levels. To study Tau pathology in more physiological conditions, we recently created Drosophila knock-in models that express either wildtype human Tau (hTauWT) or disease-associated mutant hTau (hTauV337M and hTauK369I) in place of the endogenous Drosophila Tau (dTau). Analyzing these flies as homozygotes, we could therefore detect recessive effects of the mutations while identifying dominant effects in heterozygotes. Using memory, locomotion and sleep assays, we found that homozygous mutant hTau flies showed deficits already when quite young whereas in heterozygous flies, disease phenotypes developed with aging. Homozygotes also revealed an increase in microtubule diameter, suggesting that changes in the cytoskeleton underlie the axonal degeneration we observed in these flies. In contrast, heterozygous mutant hTau flies showed abnormal axonal targeting and no detectable changes in microtubules. However, we previously showed that heterozygosity for hTauV337M interfered with synaptic homeostasis in central pacemaker neurons and we now show that heterozygous hTauK369I flies have decreased levels of proteins involved in the release of synaptic vesicles. Taken together, our results demonstrate that both mutations induce a combination of dominant and recessive disease-related phenotypes that provide behavioral and molecular insights into the etiology of Tauopathies.

Discovery and Visualization of Age-Dependent Patterns in the Diurnal Transcriptome of Drosophila.

Many critical life processes are regulated by input from 24-hour external light/dark cycles, such as metabolism, cellular homeostasis, and detoxification. The circadian clock, which helps coordinate the response to these diurnal light/dark cycles, remains rhythmic across lifespan; however, rhythmic transcript expression is altered during normal aging. To better understand how aging impacts diurnal expression, we present an improved Fourier-based method for detecting and visualizing rhythmicity that is based on the relative power of the 24-hour period compared to other periods (RP24). We apply RP24 to transcript-level expression profiles from the heads of young (5-day) and old (55-day) Drosophila melanogaster, and reveal novel age-dependent rhythmicity changes that may be masked at the gene level. We show that core clock transcripts phase advance during aging, while most rhythmic transcripts phase delay. Transcripts rhythmic only in young flies tend to peak before lights on, while transcripts only rhythmic in old peak after lights on. We show that several pathways, including glutathione metabolism, gain or lose coordinated rhythmic expression with age, providing insight into possible mechanisms of age-onset neurodegeneration. Remarkably, we find that many pathways show very robust coordinated rhythms across lifespan, highlighting their putative roles in promoting neural health. We investigate statistically enriched transcription factor binding site motifs that may be involved in these rhythmicity changes.

Manipulations of amyloid precursor protein cleavage disrupt the circadian clock in aging Drosophila.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by severe cognitive deterioration. While causes of AD pathology are debated, a large body of evidence suggests that increased cleavage of Amyloid Precursor Protein (APP) producing the neurotoxic Amyloid-ß (Aß) peptide plays a fundamental role in AD pathogenesis. One of the detrimental behavioral symptoms commonly associated with AD is the fragmentation of sleep-activity cycles with increased nighttime activity and daytime naps in humans. Sleep-activity cycles, as well as physiological and cellular rhythms, which may be important for neuronal homeostasis, are generated by a molecular system known as the circadian clock. Links between AD and the circadian system are increasingly evident but not well understood. Here we examined whether genetic manipulations of APP-like (APPL) protein cleavage in Drosophila melanogaster affect rest-activity rhythms and core circadian clock function in this model organism. We show that the increased ß-cleavage of endogenous APPL by the ß-secretase (dBACE) severely disrupts circadian behavior and leads to reduced expression of clock protein PER in central clock neurons of aging flies. Our data suggest that behavioral rhythm disruption is not a product of APPL-derived Aß production but rather may be caused by a mechanism common to both α and ß-cleavage pathways. Specifically, we show that increased production of the endogenous Drosophila Amyloid Intracellular Domain (dAICD) caused disruption of circadian rest-activity rhythms, while flies overexpressing endogenous APPL maintained stronger circadian rhythms during aging. In summary, our study offers a novel entry point toward understanding the mechanism of circadian rhythm disruption in Alzheimer's disease.

Loss of circadian clock accelerates aging in neurodegeneration-prone mutants.

Circadian clocks generate rhythms in molecular, cellular, physiological, and behavioral processes. Recent studies suggest that disruption of the clock mechanism accelerates organismal senescence and age-related pathologies in mammals. Impaired circadian rhythms are observed in many neurological diseases; however, it is not clear whether loss of rhythms is the cause or result of neurodegeneration, or both. To address this important question, we examined the effects of circadian disruption in Drosophila melanogaster mutants that display clock-unrelated neurodegenerative phenotypes. We combined a null mutation in the clock gene period (per(01)) that abolishes circadian rhythms, with a hypomorphic mutation in the carbonyl reductase gene sniffer (sni(1)), which displays oxidative stress induced neurodegeneration. We report that disruption of circadian rhythms in sni(1) mutants significantly reduces their lifespan compared to single mutants. Shortened lifespan in double mutants was coupled with accelerated neuronal degeneration evidenced by vacuolization in the adult brain. In addition, per(01)sni(1) flies showed drastically impaired vertical mobility and increased accumulation of carbonylated proteins compared to age-matched single mutant flies. Loss of per function does not affect sni mRNA expression, suggesting that these genes act via independent pathways producing additive effects. Finally, we show that per(01) mutation accelerates the onset of brain pathologies when combined with neurodegeneration-prone mutation in another gene, swiss cheese (sws(1)), which does not operate through the oxidative stress pathway. Taken together, our data suggest that the period gene may be causally involved in neuroprotective pathways in aging Drosophila.

Age-dependent effects of blue light exposure on lifespan, neurodegeneration, and mitochondria physiology in Drosophila melanogaster.

Blue light is a predominant component of light emitting devices (LEDs), which are increasingly present in our environment. There is already accumulating evidence that blue light exposure causes damage to retinal cells in vitro and in vivo; however, much less is known about potential effects of blue light on non-retinal cells. That blue light may be detrimental at the organismal level independent from retinal effect was recently shown by findings that it reduces lifespan in worms and also in flies with genetically ablated retinas. Here, we investigated the effects of blue light exposure across the fly lifespan and found that susceptibility to blue light stress is strongly age-dependent. The blue light of the same intensity and duration reduced survival and increased neurodegeneration more significantly in old flies than in young flies. These differences appear to be caused, at least in part, by impairments of mitochondrial respiratory function. We report that blue light significantly reduces the activity of Complex II in the electron transport system and decrease the biochemical activity of succinate dehydrogenase in both young and old flies. In addition, complex I and complex IV activities are reduced by age, as are ATP levels. We therefore propose that older flies are more sensitive to blue light because the light-induced mitochondrial damage potentiates the age-related impairments in energy metabolism that occurs even in darkness. Taken together, our results show that damaging effects of blue light at the organismal level are strongly age dependent and are associated with reduced activity of specific components of energy producing pathways in mitochondria.

Chronic blue light leads to accelerated aging in Drosophila by impairing energy metabolism and neurotransmitter levels.

Blue light (BL) is becoming increasingly prevalent in artificial illumination, raising concerns about its potential health hazard to humans. In fact, there is evidence suggesting that acute BL exposure may lead to oxidative stress and death of retinal cells specialized for photoreception. On the other hand, recent studies in Drosophila melanogaster demonstrated that chronic BL exposure across lifespan leads to accelerated aging manifested in reduced lifespan and brain neurodegeneration even in flies with genetically ablated eyes, suggesting that BL can damage cells and tissues not specialized for light perception. At the physiological level, BL exposure impairs mitochondria function in flies, but the metabolic underpinnings of these effects have not been studied. Here, we investigated effects of chronic BL on metabolic pathways in heads of eyes absent (eya 2 ) mutant flies in order to focus on extra-retinal tissues. We compared metabolomic profiles in flies kept for 10 or 14 days in constant BL or constant darkness, using LC-MS and GC-MS. Data analysis revealed significant alterations in the levels of several metabolites suggesting that critical cellular pathways are impacted in BL-exposed flies. In particular, dramatic metabolic rearrangements are observed in heads of flies kept in BL for 14 days, including highly elevated levels of succinate but reduced levels of pyruvate and citrate, suggesting impairments in energy production. These flies also show onset of neurodegeneration and our analysis detected significantly reduced levels of several neurotransmitters including glutamate and Gamma-aminobutyric acid (GABA), suggesting that BL disrupts brain homeostasis. Taken together, these data provide novel insights into the mechanisms by which BL interferes with vital metabolic pathways that are conserved between fly and human cells.

Circadian clock and output genes are rhythmically expressed in extratesticular ducts and accessory organs of mice.

Circadian clocks regulate multiple rhythms in mammalian tissues. In most organs core clock gene expression is oscillatory, with negative components Per and Cry peaking in antiphase to Bmal1. A notable exception is the testis, where clock genes seem nonrhythmic. Earlier mammalian studies, however, did not examine clock expression patterns in accessory ductal tissue required for sperm maturation and transport. Previous studies in insects demonstrated control of sperm maturation in vas deferens by a local circadian system. Sperm ducts express clock genes and display circadian pH changes controlled by vacuolar-type H(+)-ATPase and carbonic anhydrase (CA-II). It is unknown whether sperm-processing rhythms are conserved beyond insects. To address this question in mice housed in a light-dark environment, we examined temporal patterns of mPer1 and Bmal1 gene expression and protein abundance in epididymis, vas deferens, seminal vesicles, and prostate. Results demonstrate variable tissue-specific patterns of expression of the two genes, with variations in levels of clock proteins and their nucleo-cytoplasmic cycling observed among examined tissues. Strikingly, mPer1 and Bmal1 mRNA and proteins oscillate in antiphase in the prostate, with similar peak-trough patterns as observed in the suprachiasmatic nuclei, the brain's central clock. Genes encoding CA and a V-ATPase subunit, which are rhythmically expressed in sperm ducts of moths, are also rhythmic in some segments of murine sperm ducts. Our data suggest that some sperm duct segments may contain peripheral circadian systems whereas others may express clock genes in a pleiotropic manner.

RNA interference of the period gene affects the rhythm of sperm release in moths.

The period (per) gene is 1 of the core elements of the circadian clock mechanism in animals from insects to mammals. In clock cells of Drosophila melanogaster, per mRNA and PER protein oscillate in daily cycles. Consistent with the molecular clock model, PER moves to cell nuclei and acts as a repressor of positive clock elements. Homologs of per are known in many insects; however, specific roles of per in generating output rhythms are not known for most species. The aim of this article was to determine whether per is functionally involved in the circadian rhythm of sperm release in the moth, Spodoptera littoralis. In this species, as in other moths, rhythmic release of sperm bundles from the testis into the upper vas deferens occurs only in the evening, and this rhythm continues in the isolated reproductive system. S. littoralis was used to investigate the expression of per mRNA and protein in the 2 types of cells involved in sperm release: the cyst cells surrounding sperm bundles in the testes, and the barrier cells separating testicular follicles from the vas deferens. In cyst cells, PER showed a nuclear rhythm in light/dark (LD) cycles but was constitutively cytoplasmic in constant darkness (DD). In barrier cells, nuclear cycling of PER was observed in both LD and DD. To determine the role of PER in rhythmic sperm release in moths, testes-sperm duct complexes were treated in vitro with double-stranded fragments of per mRNA (dsRNA). This treatment significantly lowered per mRNA and protein in cyst cells and barrier cells and caused a delay of sperm release. These data demonstrate that a molecular oscillator involving the period gene plays an essential role in the regulation of rhythmic sperm release in this species.

Disease-Associated Mutant Tau Prevents Circadian Changes in the Cytoskeleton of Central Pacemaker Neurons.

A hallmark feature of Alzheimer's disease (AD) and other Tauopathies, like Frontotemporal Dementia with Parkinsonism linked to chromosome 17 (FTDP-17), is the accumulation of neurofibrillary tangles composed of the microtubule-associated protein Tau. As in AD, symptoms of FTDP-17 include cognitive decline, neuronal degeneration, and disruptions of sleep patterns. However, mechanisms by which Tau may lead to these disturbances in sleep and activity patterns are unknown. To identify such mechanisms, we have generated novel Drosophila Tauopathy models by replacing endogenous fly dTau with normal human Tau (hTau) or the FTDP-17 causing hTauV337M mutation. This mutation is localized in one of the microtubule-binding domains of hTau and has a dominant effect. Analyzing heterozygous flies, we found that aged hTauV337M flies show neuronal degeneration and locomotion deficits when compared to wild type or hTauWT flies. Furthermore, hTauV337M flies are hyperactive and they show a fragmented sleep pattern. These changes in the sleep/activity pattern are accompanied by morphological changes in the projection pattern of the central pacemaker neurons. These neurons show daily fluctuations in their connectivity, whereby synapses are increased during the day and reduced during sleep. Synapse formation requires cytoskeletal changes that can be detected by the accumulation of the end-binding protein 1 (EB1) at the site of synapse formation. Whereas, hTauWT flies show the normal day/night changes in EB1 accumulation, hTauV337M flies do not show this fluctuation. This suggests that hTauV337M disrupts sleep patterns by interfering with the cytoskeletal changes that are required for the synaptic homeostasis of central pacemaker neurons.

Lactate dehydrogenase expression modulates longevity and neurodegeneration in Drosophila melanogaster.

Lactate dehydrogenase (LDH) catalyzes the conversion of glycolysis-derived pyruvate to lactate. Lactate has been shown to play key roles in brain energetics and memory formation. However, lactate levels are elevated in aging and Alzheimer's disease patients, and it is not clear whether lactate plays protective or detrimental roles in these contexts. Here we show that Ldh transcript levels are elevated and cycle with diurnal rhythm in the heads of aged flies and this is associated with increased LDH protein, enzyme activity, and lactate concentrations. To understand the biological significance of increased Ldh gene expression, we genetically manipulated Ldh levels in adult neurons or glia. Overexpression of Ldh in both cell types caused a significant reduction in lifespan whereas Ldh down-regulation resulted in lifespan extension. Moreover, pan-neuronal overexpression of Ldh disrupted circadian locomotor activity rhythms and significantly increased brain neurodegeneration. In contrast, reduction of Ldh in neurons delayed age-dependent neurodegeneration. Thus, our unbiased genetic approach identified Ldh and lactate as potential modulators of aging and longevity in flies.

The circadian clock protein BMAL1 is necessary for fertility and proper testosterone production in mice.

Although it is well established that the circadian clock regulates mammalian reproductive physiology, the molecular mechanisms by which this regulation occurs are not clear. The authors investigated the reproductive capacity of mice lacking Bmal1 (Arntl, Mop3), one of the central circadian clock genes. They found that both male and female Bmal1 knockout (KO) mice are infertile. Gross and microscopic inspection of the reproductive anatomy of both sexes suggested deficiencies in steroidogenesis. Male Bmal1 KO mice had low testosterone and high luteinizing hormone serum concentrations, suggesting a defect in testicular Leydig cells. Importantly, Leydig cells rhythmically express BMAL1 protein, suggesting peripheral control of testosterone production by this clock protein. Expression of steroidogenic genes was reduced in testes and other steroidogenic tissues of Bmal1 KO mice. In particular, expression of the steroidogenic acute regulatory protein (StAR) gene and protein, which regulates the rate-limiting step of steroidogenesis, was decreased in testes from Bmal1 KO mice. A direct effect of BMAL1 on StAR expression in Leydig cells was indicated by in vitro experiments showing enhancement of StAR transcription by BMAL1. Other hormonal defects in male Bmal1 KO mice suggest that BMAL1 also has functions in reproductive physiology outside of the testis. These results enhance understanding of how the circadian clock regulates reproduction.

Daily blue-light exposure shortens lifespan and causes brain neurodegeneration in Drosophila.

Light is necessary for life, but prolonged exposure to artificial light is a matter of increasing health concern. Humans are exposed to increased amounts of light in the blue spectrum produced by light-emitting diodes (LEDs), which can interfere with normal sleep cycles. The LED technologies are relatively new; therefore, the long-term effects of exposure to blue light across the lifespan are not understood. We investigated the effects of light in the model organism, Drosophila melanogaster, and determined that flies maintained in daily cycles of 12-h blue LED and 12-h darkness had significantly reduced longevity compared with flies maintained in constant darkness or in white light with blue wavelengths blocked. Exposure of adult flies to 12 h of blue light per day accelerated aging phenotypes causing damage to retinal cells, brain neurodegeneration, and impaired locomotion. We report that brain damage and locomotor impairments do not depend on the degeneration in the retina, as these phenotypes were evident under blue light in flies with genetically ablated eyes. Blue light induces expression of stress-responsive genes in old flies but not in young, suggesting that cumulative light exposure acts as a stressor during aging. We also determined that several known blue-light-sensitive proteins are not acting in pathways mediating detrimental light effects. Our study reveals the unexpected effects of blue light on fly brain and establishes Drosophila as a model in which to investigate long-term effects of blue light at the cellular and organismal level.

Yolk protein is expressed in the insect testis and interacts with sperm.

BACKGROUND: Male and female gametes follow diverse developmental pathways dictated by their distinct roles in fertilization. While oocytes of oviparous animals accumulate yolk in the cytoplasm, spermatozoa slough off most of their cytoplasm in the process of individualization. Mammalian spermatozoa released from the testis undergo extensive modifications in the seminal ducts involving a variety of glycoproteins. Ultrastructural studies suggest that glycoproteins are involved in sperm maturation in insects; however, their characterization at the molecular level is lacking. We reported previously that the circadian clock controls sperm release and maturation in several insect species. In the moth, Spodoptera littoralis, the secretion of glycoproteins into the seminal fluid occurs in a daily rhythmic pattern. The purpose of this study was to characterize seminal fluid glycoproteins in this species and elucidate their role in the process of sperm maturation. RESULTS: We collected seminal fluid proteins from males before and after daily sperm release. These samples were separated by 2-D gel electrophoresis, and gels were treated with a glycoprotein-detecting probe. We observed a group of abundant glycoproteins in the sample collected after sperm release, which was absent in the sample collected before sperm release. Sequencing of these glycoproteins by mass spectroscopy revealed peptides bearing homology with components of yolk, which is known to accumulate in developing oocytes. This unexpected result was confirmed by Western blotting demonstrating that seminal fluid contains protein immunoreactive to antibody against yolk protein YP2 produced in the follicle cells surrounding developing oocytes. We cloned the fragment of yp2 cDNA from S. littoralis and determined that it is expressed in both ovaries and testes. yp2 mRNA and YP2 protein were detected in the somatic cyst cells enveloping sperm inside the testis. During the period of sperm release, YP2 protein appears in the seminal fluid and forms an external coat on spermatozoa. CONCLUSION: One of the yolk protein precursors YP2, which in females accumulate in the oocytes to provision developing embryos, appears to have a second male-specific role. It is produced in the testes and released into the seminal fluid where it interacts with sperm. These data reveal unexpected common factor in the maturation of insect eggs and sperm.

Circadian regulation of response to oxidative stress in Drosophila melanogaster.

Circadian rhythms are fundamental biological phenomena generated by molecular genetic mechanisms known as circadian clocks. There is increasing evidence that circadian synchronization of physiological and cellular processes contribute to the wellness of organisms, curbing pathologies such as cancer and premature aging. Therefore, there is a need to understand how circadian clocks orchestrate interactions between the organism's internal processes and the environment. Here, we explore the nexus between the clock and oxidative stress susceptibility in Drosophila melanogaster. We exposed flies to acute oxidative stress induced by hydrogen peroxide (H(2)O(2)), and determined that mortality rates were dependent on time at which exposure occurred during the day/night cycle. The daily susceptibility rhythm was abolished in flies with a null mutation in the core clock gene period (per) abrogating clock function. Furthermore, lack of per increased susceptibility to H(2)O(2) compared to wild-type flies, coinciding with enhanced generation of mitochondrial H(2)O(2) and decreased catalase activity due to oxidative damage. Taken together, our data suggest that the circadian clock gene period is essential for maintaining a robust anti-oxidative defense.

Circadian regulation of metabolism and healthspan in Drosophila.

Circadian clocks generate daily rhythms in gene expression, cellular functions, physiological processes and behavior. The core clock mechanism consists of transcriptional-translational negative feedback loops that turn over with an endogenous circa 24h period. Classical genetic experiments in the fly Drosophila melanogaster played an essential role in identification of clock genes that turned out to be largely conserved between flies and mammals. Like in mammals, circadian clocks in flies generate transcriptional rhythms in a variety of metabolic pathways related to feeding and detoxification. Given that rhythms pervade metabolism and the loss of metabolic homeostasis is involved in aging and disease, there is increasing interest in understanding how the clocks and the rhythms they control change during aging. The importance of circadian clocks for healthy aging is supported by studies reporting that genetic or environmental clock disruptions are associated with reduced healthspan and lifespan. For example, arrhythmia caused by mutations in core clock genes lead to symptoms of accelerated aging in both flies and mammals, including neurodegenerative phenotypes. Despite the wealth of descriptive data, the mechanisms by which functional clocks confer healthspan and lifespan benefits are poorly understood. Studies in Drosophila discussed here are beginning to unravel causative relationships between the circadian system and aging. In particular, recent data suggest that clocks may be involved in inducing rhythmic expression of specific genes late in life in response to age-related increase in oxidative stress. This review will summarize insights into links between circadian system and aging in Drosophila, which were obtained using powerful genetics tools available for this model organism and taking advantage of the short adult lifespan in flies that is measured in days rather than years.

Regulation of copulation duration by period and timeless in Drosophila melanogaster.

The circadian clock involves several clock genes encoding interacting transcriptional regulators. Mutations in clock genes in Drosophila melanogaster, period (per), timeless (tim), Clock (Clk), and cycle (cyc), produce multiple phenotypes associated with physiology, behavior, development, and morphology. It is not clear whether these genes always work as clock components or may also act in some unknown pleiotropic fashion. We report here that per and tim are involved in a novel, male-specific phenotype that affects behavioral timing on the order of minutes. Males lacking per or tim copulate significantly longer than males with normal per or tim function, while females do not show this effect. No correlation between fertility and extended copulation duration was found. Several lines of evidence suggest that the time in copula (TIC) is not regulated by the known clock mechanism. First, the period of free-running clock oscillations does not appear to affect this phenotype. Second, constant light, which abolishes the clock function, does not alter TIC. Finally, mutations in the positively acting clock transcription factors, Clk and cyc, do not affect TIC. Our study extends the repertoire of behavioral functions involving per and tim genes and uncovers another time scale over which these genes may act.

Ectopic CRYPTOCHROME renders TIM light sensitive in the Drosophila ovary.

The period (per) and timeless (tim) genes play a central role in the Drosophila circadian clock mechanism. PERIOD (PER) and TIMELESS (TIM) proteins periodically accumulate in the nuclei of pace-making cells in the fly brain and many cells in peripheral organs. In contrast, TIM and PER in the ovarian follicle cells remain cytoplasmic and do not show daily oscillations in their levels. Moreover, TIM is not light sensitive in the ovary, while it is highly sensitive to this input in circadian tissues. The mechanism underlying this intriguing difference is addressed here. It is demonstrated that the circadian photoreceptor CRYPTOCHROME (CRY) is not expressed in ovarian tissues. Remarkably, ectopic cry expression in the ovary is sufficient to cause degradation of TIM after exposure to light. In addition, PER levels are reduced in response to light when CRY is present, as observed in circadian cells. Hence, CRY is the key component of the light input pathway missing in the ovary. However, the factors regulating PER and TIM levels downstream of light/cry action appear to be present in this non-circadian organ.

Age-Related Changes in the Expression of the Circadian Clock Protein PERIOD in Drosophila Glial Cells.

Circadian clocks consist of molecular negative feedback loops that coordinate physiological, neurological, and behavioral variables into "circa" 24-h rhythms. Rhythms in behavioral and other circadian outputs tend to weaken during aging, as evident in progressive disruptions of sleep-wake cycles in aging organisms. However, less is known about the molecular changes in the expression of clock genes and proteins that may lead to the weakening of circadian outputs. Western blot studies have demonstrated that the expression of the core clock protein PERIOD (PER) declines in the heads of aged Drosophila melanogaster flies. This age-related decline in PER does not occur in the central pacemaker neurons but has been demonstrated so far in retinal photoreceptors. Besides photoreceptors, clock proteins are also expressed in fly glia, which play important roles in neuronal homeostasis and are further categorized into subtypes based on morphology and function. While previous studies of mammalian glial cells have demonstrated the presence of functional clocks in astrocytes and microglia, it is not known which glial cell types in Drosophila express clock proteins and how their expression may change in aged individuals. Here, we conducted immunocytochemistry experiments to identify which glial subtypes express PER protein suggestive of functional circadian clocks. Glial cell subtypes that showed night-time accumulation and day-time absence in PER consistent with oscillations reported in the pacemaker neurons were selected to compare the level of PER protein between young and old flies. Our data demonstrate that some glial subtypes show rhythmic PER expression and the relative PER levels become dampened with advanced age. Identification of glial cell types that display age-related dampening of PER levels may help to understand the cellular changes that contribute to the loss of homeostasis in the aging brain.

Circadian deep sequencing reveals stress-response genes that adopt robust rhythmic expression during aging.

Disruption of the circadian clock, which directs rhythmic expression of numerous output genes, accelerates aging. To enquire how the circadian system protects aging organisms, here we compare circadian transcriptomes in heads of young and old Drosophila melanogaster. The core clock and most output genes remained robustly rhythmic in old flies, while others lost rhythmicity with age, resulting in constitutive over- or under-expression. Unexpectedly, we identify a subset of genes that adopted increased or de novo rhythmicity during aging, enriched for stress-response functions. These genes, termed late-life cyclers, were also rhythmically induced in young flies by constant exposure to exogenous oxidative stress, and this upregulation is CLOCK-dependent. We also identify age-onset rhythmicity in several putative primary piRNA transcripts overlapping antisense transposons. Our results suggest that, as organisms age, the circadian system shifts greater regulatory priority to the mitigation of accumulating cellular stress.

Circadian rhythm in mRNA expression of the glutathione synthesis gene Gclc is controlled by peripheral glial clocks in Drosophila melanogaster.

Circadian coordination of metabolism, physiology, and behaviour is found in all living kingdoms. Clock genes are transcriptional regulators, and their rhythmic activities generate daily rhythms in clock-controlled genes which result in cellular and organismal rhythms. Insects provide numerous examples of rhythms in behaviour and reproduction, but less is known about control of metabolic processes by circadian clocks in insects. Recent data suggest that several pathways involved in protecting cells from oxidative stress may be modulated by the circadian system, including genes involved in glutathione (GSH) biosynthesis. Specifically, rhythmic expression of the gene encoding the catalytic subunit (Gclc) of the rate-limiting GSH biosynthetic enzyme was detected in Drosophila melanogaster heads. The aim of this study was to determine which clocks in the fly multi-oscillatory circadian system are responsible for Gclc rhythms. Genetic disruption of tissue-specific clocks in D. melanogaster revealed that transcriptional rhythms in Gclc mRNA levels occur independently of the central pacemaker neurons, because these rhythms persisted in heads of behaviourally arrhythmic flies with a disabled central clock but intact peripheral clocks. Disrupting the clock specifically in glial cells abolished rhythmic expression of Gclc, suggesting that glia play an important role in Gclc transcriptional regulation, which may contribute to maintaining homeostasis in the fly nervous system.