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.

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.

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.

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.

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.

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.

Genetic and biochemical strategies for identifying Drosophila genes that function in circadian control.

Explicit biochemical models have been elaborated for the circadian oscillators of cyanobacterial, fungal, insect, and mammalian species. In contrast, much remains to be learned about how such circadian oscillators regulate rhythmic physiological processes. This article summarizes contemporary genetic and biochemical strategies that are useful for identifying gene products that have a role in circadian control.

Structure and absolute stereochemistry of phormidolide, a new toxic metabolite from the marine cyanobacterium Phormidium sp.

The extract from a laboratory culture of an Indonesian isolate of the cyanobacterium Phormidium sp. displayed inhibitory activity in a Ras-Raf protein interaction assay. Assay-guided fractionation led to the isolation of both active and inactive materials of novel structure. The major inactive metabolite, phormidolide, was nevertheless highly toxic to brine shrimp (LC(50) = 1.5 microM), and hence, its structure was elucidated using various spectroscopic methods, primarily NMR. A series of partial structures were developed from standard experiments and then assembled using GHMBC, 2D INADEQUATE, and ACCORD-ADEQUATE data obtained on a (13)C-enriched sample. The relative stereochemistry at phormidolide's 11 chiral centers was established using the J-based configuration analysis method in concert with the G-BIRD(R)-HSQMBC NMR experiment. Absolute stereochemistry was determined on a bis-acetonide derivative using the variable temperature Mosher ester method. The robust number of NMR restraints provided from determination of most homonuclear and heteronuclear coupling constants in phormidolide, along with an abundance of NOE information, allowed construction of a refined lowest energy three-dimensional structure in Macromodel. Phormidolide is one of only a few macrolide-type natural products to be reported from marine cyanobacteria.

Cell-specific expression of the lark RNA-binding protein in Drosophila results in morphological and circadian behavioral phenotypes.

Past studies have implicated the Drosophila LARK protein in the circadian control of adult eclosion behavior. LARK has a broad tissue pattern of distribution, and is pan-neuronal in the differentiated brain. In certain peptidergic neurons, LARK abundance changes in a circadian manner. However, the precise cellular requirement for LARK, with respect to circadian behavior, is still not known. To explore this issue, we employed the GAL4/UAS binary expression system to increase LARK abundance in defined neuronal cell types. Interestingly, LARK expression in Crustacean Cardioactive Peptide (CCAP) neurons caused an early-eclosion phenotype, whereas a similar perturbation in the Eclosion Hormone (EH) cells resulted in abnormally late peaks of eclosion. Surprisingly, LARK expression in Pigment Dispersing Factor (PDF)- or TIMELESS (TIM)-containing clock neurons caused behavioral arrhythmicity, even though clock protein cycling was found to be normal in these flies. Although the observed effects of LARK expression mirrored those seen with genetic ablation of the relevant peptidergic populations, there was no evidence of defective cell development or morphology. This suggests that an alteration of cell function rather than cell death is the cause of the aberrant phenotypes. Diminished PDF immunoreactivity in flies expressing LARK in the PDF neurons suggests that an effect on neuropeptide synthesis, transport, or release may contribute to the observed arrhythmicity. Importantly, the expression of LARK in several other cell populations did not have detectable effects on development, viability or behavior, indicating a specificity of action within certain cell types.