Drosophila noktochor regulates night sleep via a local mushroom body circuit.

We show that a sleep-regulating, Ig-domain protein (NKT) is secreted from Drosophila mushroom body (MB) α'/ß' neurons to act locally on other MB cell types. Pan-neuronal or broad MB expression of membrane-tethered NKT (tNkt) protein reduced sleep, like that of an NKT null mutant, suggesting blockade of a receptor mediating endogenous NKT action. In contrast, expression in neurons requiring NKT (the MB α'/ß' cells), or non-MB sleep-regulating centers, did not reduce night sleep, indicating the presence of a local MB sleep-regulating circuit consisting of communicating neural subtypes. We suggest that the leucocyte-antigen-related like (Lar) transmembrane receptor may mediate NKT action. Knockdown or overexpression of Lar in the MB increased or decreased sleep, respectively, indicating the receptor promotes wakefulness. Surprisingly, selective expression of tNkt or knockdown of Lar in MB wake-promoting cells increased rather than decreased sleep, suggesting that NKT acts on wake- as well as sleep-promoting cell types to regulate sleep.

Circadian regulation of the Drosophila astrocyte transcriptome.

Recent studies have demonstrated that astrocytes cooperate with neurons of the brain to mediate circadian control of many rhythmic processes including locomotor activity and sleep. Transcriptional profiling studies have described the overall rhythmic landscape of the brain, but few have employed approaches that reveal heterogeneous, cell-type specific rhythms of the brain. Using cell-specific isolation of ribosome-bound RNAs in Drosophila, we constructed the first circadian "translatome" for astrocytes. This analysis identified 293 "cycling genes" in astrocytes, most with mammalian orthologs. A subsequent behavioral genetic screen identified a number of genes whose expression is required in astrocytes for normal sleep behavior. In particular, we show that certain genes known to regulate fly innate immune responses are also required for normal sleep patterns.

Regulation of rhythmic behaviors by astrocytes.

Glial astrocytes of vertebrates and invertebrates are important modulators of nervous system development, physiology, and behavior. In all species examined, astrocytes of the adult brain contain conserved circadian clocks, and multiple studies have shown that these glial cells participate in the regulation of circadian behavior and sleep. This short review summarizes recent work, using fruit fly (Drosophila) and mouse models, that document participation of astrocytes and their endogenous circadian clocks in the control of rhythmic behavior. This article is categorized under: Gene Expression and Transcriptional Hierarchies > Regulatory Mechanisms Nervous System Development > Flies.

A Secreted Ig-Domain Protein Required in Both Astrocytes and Neurons for Regulation of Drosophila Night Sleep.

Endogenous rhythmic behaviors are evolutionarily conserved and essential for life. In mammalian and invertebrate models, well-characterized neuronal circuits and evolutionarily conserved mechanisms regulate circadian behavior and sleep [1-4]. In Drosophila, neuronal populations located in multiple brain regions mediate arousal, sleep drive, and homeostasis (reviewed in [3, 5-7]). Similar to mammals [8], there is also evidence that fly glial cells modulate the neuronal circuits controlling rhythmic behaviors, including sleep [1]. Here, we describe a novel gene (CG14141; aka Nkt) that is required for normal sleep. NKT is a 162-amino-acid protein with a single IgC2 immunoglobulin (Ig) domain and a high-quality signal peptide [9], and we show evidence that it is secreted, similar to its C. elegans ortholog (OIG-4) [10]. We demonstrate that Nkt-null flies or those with selective knockdown in either neurons or glia have decreased and fragmented night sleep, indicative of a non-redundant requirement in both cell types. We show that Nkt is required in fly astrocytes and in a specific set of wake-promoting neurons-the mushroom body (MB) α'ß' cells that link sleep to memory consolidation [11]. Importantly, Nkt gene expression is required in the adult nervous system for normal sleep, consistent with a physiological rather than developmental function for the Ig-domain protein.

Regulation of Circadian Behavior by Astroglial MicroRNAs in Drosophila.

We describe a genome-wide microRNA (miRNA)-based screen to identify brain glial cell functions required for circadian behavior. To identify glial miRNAs that regulate circadian rhythmicity, we employed a collection of "miR-sponges" to inhibit miRNA function in a glia-specific manner. Our initial screen identified 20 glial miRNAs that regulate circadian behavior. We studied two miRNAs, miR-263b and miR-274, in detail and found that both function in adult astrocytes to regulate behavior. Astrocyte-specific inhibition of miR-263b or miR-274 in adults acutely impairs circadian locomotor activity rhythms with no effect on glial or clock neuronal cell viability. To identify potential RNA targets of miR-263b and miR-274, we screened 35 predicted miRNA targets, employing RNA interference-based approaches. Glial knockdown of two putative miR-274 targets, CG4328 and MESK2, resulted in significantly decreased rhythmicity. Homology of the miR-274 targets to mammalian counterparts suggests mechanisms that might be relevant for the glial regulation of rhythmicity.

TRAP-seq Profiling and RNAi-Based Genetic Screens Identify Conserved Glial Genes Required for Adult Drosophila Behavior.

Although, glial cells have well characterized functions in the developing and mature brain, it is only in the past decade that roles for these cells in behavior and plasticity have been delineated. Glial astrocytes and glia-neuron signaling, for example, are now known to have important modulatory functions in sleep, circadian behavior, memory and plasticity. To better understand mechanisms of glia-neuron signaling in the context of behavior, we have conducted cell-specific, genome-wide expression profiling of adult Drosophila astrocyte-like brain cells and performed RNA interference (RNAi)-based genetic screens to identify glial factors that regulate behavior. Importantly, our studies demonstrate that adult fly astrocyte-like cells and mouse astrocytes have similar molecular signatures; in contrast, fly astrocytes and surface glia-different classes of glial cells-have distinct expression profiles. Glial-specific expression of 653 RNAi constructs targeting 318 genes identified multiple factors associated with altered locomotor activity, circadian rhythmicity and/or responses to mechanical stress (bang sensitivity). Of interest, 1 of the relevant genes encodes a vesicle recycling factor, 4 encode secreted proteins and 3 encode membrane transporters. These results strongly support the idea that glia-neuron communication is vital for adult behavior.

The ROP vesicle release factor is required in adult Drosophila glia for normal circadian behavior.

We previously showed that endocytosis and/or vesicle recycling mechanisms are essential in adult Drosophila glial cells for the neuronal control of circadian locomotor activity. In this study, our goal was to identify specific glial vesicle trafficking, recycling, or release factors that are required for rhythmic behavior. From a glia-specific, RNAi-based genetic screen, we identified eight glial factors that are required for normally robust circadian rhythms in either a light-dark cycle or in constant dark conditions. In particular, we show that conditional knockdown of the ROP vesicle release factor in adult glial cells results in arrhythmic behavior. Immunostaining for ROP reveals reduced protein in glial cell processes and an accumulation of the Par Domain Protein 1ε (PDP1ε) clock output protein in the small lateral clock neurons. These results suggest that glia modulate rhythmic circadian behavior by secretion of factors that act on clock neurons to regulate a clock output factor.

Comparison of larval and adult Drosophila astrocytes reveals stage-specific gene expression profiles.

The analysis of adult astrocyte glial cells has revealed a remarkable heterogeneity with regard to morphology, molecular signature, and physiology. A key question in glial biology is how such heterogeneity arises during brain development. One approach to this question is to identify genes with differential astrocyte expression during development; certain genes expressed later in neural development may contribute to astrocyte differentiation. We have utilized the Drosophila model and Translating Ribosome Affinity Purification (TRAP)-RNA-seq methods to derive the genome-wide expression profile of Drosophila larval astrocyte-like cells (hereafter referred to as astrocytes) for the first time. These studies identified hundreds of larval astrocyte-enriched genes that encode proteins important for metabolism, energy production, and protein synthesis, consistent with the known role of astrocytes in the metabolic support of neurons. Comparison of the larval profile with that observed for adults has identified genes with astrocyte-enriched expression specific to adulthood. These include genes important for metabolism and energy production, translation, chromatin modification, protein glycosylation, neuropeptide signaling, immune responses, vesicle-mediated trafficking or secretion, and the regulation of behavior. Among these functional classes, the expression of genes important for chromatin modification and vesicle-mediated trafficking or secretion is overrepresented in adult astrocytes based on Gene Ontology analysis. Certain genes with selective adult enrichment may mediate functions specific to this stage or may be important for the differentiation or maintenance of adult astrocytes, with the latter perhaps contributing to population heterogeneity.

Glial cell regulation of rhythmic behavior.

Brain glial cells, in particular astrocytes and microglia, secrete signaling molecules that regulate glia-glia or glia-neuron communication and synaptic activity. While much is known about roles of glial cells in nervous system development, we are only beginning to understand the physiological functions of such cells in the adult brain. Studies in vertebrate and invertebrate models, in particular mice and Drosophila, have revealed roles of glia-neuron communication in the modulation of complex behavior. This chapter emphasizes recent evidence from studies of rodents and Drosophila that highlight the importance of glial cells and similarities or differences in the neural circuits regulating circadian rhythms and sleep in the two models. The chapter discusses cellular, molecular, and genetic approaches that have been useful in these models for understanding how glia-neuron communication contributes to the regulation of rhythmic behavior.

Translational regulation of the DOUBLETIME/CKIδ/ε kinase by LARK contributes to circadian period modulation.

The Drosophila homolog of Casein Kinase I δ/ε, DOUBLETIME (DBT), is required for Wnt, Hedgehog, Fat and Hippo signaling as well as circadian clock function. Extensive studies have established a critical role of DBT in circadian period determination. However, how DBT expression is regulated remains largely unexplored. In this study, we show that translation of dbt transcripts are directly regulated by a rhythmic RNA-binding protein (RBP) called LARK (known as RBM4 in mammals). LARK promotes translation of specific alternative dbt transcripts in clock cells, in particular the dbt-RC transcript. Translation of dbt-RC exhibits circadian changes under free-running conditions, indicative of clock regulation. Translation of a newly identified transcript, dbt-RE, is induced by light in a LARK-dependent manner and oscillates under light/dark conditions. Altered LARK abundance affects circadian period length, and this phenotype can be modified by different dbt alleles. Increased LARK delays nuclear degradation of the PERIOD (PER) clock protein at the beginning of subjective day, consistent with the known role of DBT in PER dynamics. Taken together, these data support the idea that LARK influences circadian period and perhaps responses of the clock to light via the regulated translation of DBT. Our study is the first to investigate translational control of the DBT kinase, revealing its regulation by LARK and a novel role of this RBP in Drosophila circadian period modulation.

Translational profiling of clock cells reveals circadianly synchronized protein synthesis.

Genome-wide studies of circadian transcription or mRNA translation have been hindered by the presence of heterogeneous cell populations in complex tissues such as the nervous system. We describe here the use of a Drosophila cell-specific translational profiling approach to document the rhythmic "translatome" of neural clock cells for the first time in any organism. Unexpectedly, translation of most clock-regulated transcripts--as assayed by mRNA ribosome association--occurs at one of two predominant circadian phases, midday or mid-night, times of behavioral quiescence; mRNAs encoding similar cellular functions are translated at the same time of day. Our analysis also indicates that fundamental cellular processes--metabolism, energy production, redox state (e.g., the thioredoxin system), cell growth, signaling and others--are rhythmically modulated within clock cells via synchronized protein synthesis. Our approach is validated by the identification of mRNAs known to exhibit circadian changes in abundance and the discovery of hundreds of novel mRNAs that show translational rhythms. This includes Tdc2, encoding a neurotransmitter synthetic enzyme, which we demonstrate is required within clock neurons for normal circadian locomotor activity.

Dispensable, redundant, complementary, and cooperative roles of dopamine, octopamine, and serotonin in Drosophila melanogaster.

To investigate the regulation of Drosophila melanogaster behavior by biogenic amines, we have exploited the broad requirement of the vesicular monoamine transporter (VMAT) for the vesicular storage and exocytotic release of all monoamine neurotransmitters. We used the Drosophila VMAT (dVMAT) null mutant to globally ablate exocytotic amine release and then restored DVMAT activity in either individual or multiple aminergic systems, using transgenic rescue techniques. We find that larval survival, larval locomotion, and female fertility rely predominantly on octopaminergic circuits with little apparent input from the vesicular release of serotonin or dopamine. In contrast, male courtship and fertility can be rescued by expressing DVMAT in octopaminergic or dopaminergic neurons, suggesting potentially redundant circuits. Rescue of major aspects of adult locomotion and startle behavior required octopamine, but a complementary role was observed for serotonin. Interestingly, adult circadian behavior could not be rescued by expression of DVMAT in a single subtype of aminergic neurons, but required at least two systems, suggesting the possibility of unexpected cooperative interactions. Further experiments using this model will help determine how multiple aminergic systems may contribute to the regulation of other behaviors. Our data also highlight potential differences between behaviors regulated by standard exocytotic release and those regulated by other mechanisms.

Development of dendrite polarity in Drosophila neurons.

BACKGROUND: Drosophila neurons have dendrites that contain minus-end-out microtubules. This microtubule arrangement is different from that of cultured mammalian neurons, which have mixed polarity microtubules in dendrites. RESULTS: To determine whether Drosophila and mammalian dendrites have a common microtubule organization during development, we analyzed microtubule polarity in Drosophila dendritic arborization neuron dendrites at different stages of outgrowth from the cell body in vivo. As dendrites initially extended, they contained mixed polarity microtubules, like mammalian neurons developing in culture. Over a period of several days this mixed microtubule array gradually matured to a minus-end-out array. To determine whether features characteristic of dendrites were localized before uniform polarity was attained, we analyzed dendritic markers as dendrites developed. In all cases the markers took on their characteristic distribution while dendrites had mixed polarity. An axonal marker was also quite well excluded from dendrites throughout development, although this was perhaps more efficient in mature neurons. To confirm that dendrite character could be acquired in Drosophila while microtubules were mixed, we genetically disrupted uniform dendritic microtubule organization. Dendritic markers also localized correctly in this case. CONCLUSIONS: We conclude that developing Drosophila dendrites initially have mixed microtubule polarity. Over time they mature to uniform microtubule polarity. Dendrite identity is established before the mature microtubule arrangement is attained, during the period of mixed microtubule polarity.

Cellular requirements for LARK in the Drosophila circadian system.

RNA-binding proteins mediate posttranscriptional functions in the circadian systems of multiple species. A conserved RNA recognition motif (RRM) protein encoded by the lark gene is postulated to serve circadian output and molecular oscillator functions in Drosophila and mammals, respectively. In no species, however, has LARK been eliminated, in vivo, to determine the consequences for circadian timing. The present study utilized RNA interference (RNAi) techniques in Drosophila to decrease LARK levels in clock neurons and other cell types in order to evaluate the circadian functions of the protein. Knockdown of LARK in timeless (TIM)- or pigment dispersing factor (PDF)-containing clock cells caused a significant number of flies to exhibit arrhythmic locomotor activity, demonstrating a requirement for the protein in pacemaker cells. There was no obvious effect on PER protein cycling in lark interference (RNAi) flies, but a knockdown within the PDF neurons was associated with increased PDF immunoreactivity at the dorsal termini of the small ventral lateral neuronal (s-LNv) projections, suggesting an effect on neuropeptide release. The expression of lark RNAi in multiple neurosecretory cell populations demonstrated that LARK is required within pacemaker and nonpacemaker cells for the manifestation of normal locomotor activity rhythms. Interestingly, decreased LARK function in the prothoracic gland (PG), a peripheral organ containing a clock required for the circadian control of eclosion, was associated with weak population eclosion rhythms or arrhythmicity.

The C-terminal kinase and ERK-binding domains of Drosophila S6KII (RSK) are required for phosphorylation of the protein and modulation of circadian behavior.

A detailed structure/function analysis of Drosophila p90 ribosomal S6 kinase (S6KII) or its mammalian homolog RSK has not been performed in the context of neuronal plasticity or behavior. We previously reported that S6KII is required for normal circadian periodicity. Here we report a site-directed mutagenesis of S6KII and analysis of mutants, in vivo, that identifies functional domains and phosphorylation sites critical for the regulation of circadian period. We demonstrate, for the first time, a role for the S6KII C-terminal kinase that is independent of its known role in activation of the N-terminal kinase. Both S6KII C-terminal kinase activity and its ERK-binding domain are required for wild-type circadian period and normal phosphorylation status of the protein. In contrast, the N-terminal kinase of S6KII is dispensable for modulation of circadian period and normal phosphorylation of the protein. We also show that particular sites of S6KII phosphorylation, Ser-515 and Thr-732, are essential for normal circadian behavior. Surprisingly, the phosphorylation of S6KII residues, in vivo, does not follow a strict sequential pattern, as implied by certain cell-based studies of mammalian RSK protein.

Glial cell modulation of circadian rhythms.

Studies of Drosophila and mammals have documented circadian changes in the morphology and biochemistry of glial cells. In addition, it is known that astrocytes of flies and mammals contain evolutionarily conserved circadian molecular oscillators that are similar to neuronal oscillators. In several sections of this review, I summarize the morphological and biochemical rhythms of glia that may contribute to circadian control. I also discuss the evidence suggesting that glia-neuron interactions may be critical for circadian timing in both flies and mammals. Throughout the review, I attempt to compare and contrast findings from these invertebrate and vertebrate models so as to provide a synthesis of current knowledge and indicate potential research avenues that may be useful for better understanding the roles of glial cells in the circadian system.

Glial cells physiologically modulate clock neurons and circadian behavior in a calcium-dependent manner.

BACKGROUND: An important goal of contemporary neuroscience research is to define the neural circuits and synaptic interactions that mediate behavior. In both mammals and Drosophila, the neuronal circuitry controlling circadian behavior has been the subject of intensive investigation, but roles for glial cells in the networks controlling rhythmic behavior have only begun to be defined in recent studies. RESULTS: Here, we show that conditional, glial-specific genetic manipulations affecting membrane (vesicle) trafficking, the membrane ionic gradient, or calcium signaling lead to circadian arrhythmicity in adult behaving Drosophila. Correlated and reversible effects on a clock neuron peptide transmitter (PDF) and behavior demonstrate the capacity for glia-to-neuron signaling in the circadian circuitry. These studies also reveal the importance of a single type of glial cell-the astrocyte-and glial internal calcium stores in the regulation of circadian rhythms. CONCLUSIONS: This is the first demonstration in any system that adult glial cells can physiologically modulate circadian neuronal circuitry and behavior. A role for astrocytes and glial calcium signaling in the regulation of Drosophila circadian rhythms emphasizes the conservation of cellular and molecular mechanisms that regulate behavior in mammals and insects.

The evolutionarily conserved RNA binding protein SMOOTH is essential for maintaining normal muscle function.

The Drosophila smooth gene encodes an RNA binding protein that has been well conserved through evolution. To investigate the pleiotropic functions mediated by the smooth gene, we have selected and characterized two sm mutants, which are viable as adults yet display robust phenotypes (including a significant decrease in lifespan). Utilizing these mutants, we have made the novel observation that disruption of the smooth/CG9218 locus leads to age-dependent muscle degeneration, and motor dysfunction. Histological characterization of adult sm mutants revealed marked abnormalities in the major thoracic tubular muscle: the tergal depressor of the trochanter (TDT). Corresponding defects include extensive loss/disruption of striations and nuclei. These pathological changes are recapitulated in flies that express a smooth RNA interference construct (sm RNAi) in the mesoderm. In contrast, targeting sm RNAi constructs to motor neurons does not alter muscle morphology. In addition to examining the TDT phenotype, we explored whether other muscular abnormalities were evident. Utilizing physiological assays developed in the laboratory, we have found that the thoracic muscle defect is preceded by dysmotility of the gastrointestinal tract. SMOOTH thus joins a growing list of hnRNPs that have previously been linked to muscle physiology/pathophysiology. Our findings in Drosophila set the stage for investigating the role of the corresponding mammalian homolog, hnRNP L, in muscle function.

Altered LARK expression perturbs development and physiology of the Drosophila PDF clock neurons.

The LARK RNA-binding protein (RBP) has well documented roles in the circadian systems of Drosophila and mammals. Recent studies have demonstrated that the Drosophila LARK RBP is associated with many mRNA targets, in vivo, including those that regulate either neurophysiology or development of the nervous system. In the present study, we have employed conditional expression techniques to distinguish developmental and physiological functions of LARK for a defined class of neurons: the Pigment-Dispersing Factor (PDF)-containing LNv clock neurons. We found that increased LARK expression during development dramatically alters the small LNv class of neurons with no obvious effects on the large LNv cells. Conversely, conditional expression of LARK at the adult stage results in altered clock protein rhythms and circadian locomotor activity, even though neural morphology is normal in such animals. Electrophysiological analyses at the larval neuromuscular junction indicate a role for LARK in regulating neuronal excitability. Altogether, our results demonstrate that LARK activity is critical for neuronal development and physiology.

Ribosomal s6 kinase cooperates with casein kinase 2 to modulate the Drosophila circadian molecular oscillator.

There is a universal requirement for post-translational regulatory mechanisms in circadian clock systems. Previous work in Drosophila has identified several kinases, phosphatases, and an E3 ligase that are critical for determining the nuclear translocation and/or stability of clock proteins. The present study evaluated the function of p90 ribosomal S6 kinase (RSK) in the Drosophila circadian system. In mammals, RSK1 is a light- and clock-regulated kinase known to be activated by the mitogen-activated protein kinase pathway, but there is no direct evidence that it functions as a component of the circadian system. Here, we show that Drosophila S6KII RNA displays rhythms in abundance, indicative of circadian control. Importantly, an S6KII null mutant exhibits a short-period circadian phenotype that can be rescued by expression of the wild-type gene in clock neurons, indicating a role for S6KII in the molecular oscillator. Peak PER clock protein expression is elevated in the mutant, indicative of enhanced stability, whereas per mRNA level is decreased, consistent with enhanced feedback repression. Gene reporter assays show that decreased S6KII is associated with increased PER repression. Surprisingly, we demonstrate a physical interaction between S6KII and the casein kinase 2 regulatory subunit (CK2beta), suggesting a functional relationship between the two kinases. In support of such a relationship, there are genetic interactions between S6KII and CK2 mutations, in vivo, which indicate that CK2 activity is required for S6KII action. We propose that the two kinases cooperate within clock neurons to fine-tune circadian period, improving the precision of the clock mechanism.