orsai, the Drosophila homolog of human ETFRF1, links lipid catabolism to growth control.

BACKGROUND: Lipid homeostasis is an evolutionarily conserved process that is crucial for energy production, storage and consumption. Drosophila larvae feed continuously to achieve the roughly 200-fold increase in size and accumulate sufficient reserves to provide all energy and nutrients necessary for the development of the adult fly. The mechanisms controlling this metabolic program are poorly understood. RESULTS: Herein we identified a highly conserved gene, orsai (osi), as a key player in lipid metabolism in Drosophila. Lack of osi function in the larval fat body, the regulatory hub of lipid homeostasis, reduces lipid reserves and energy output, evidenced by decreased ATP production and increased ROS levels. Metabolic defects due to reduced Orsai (Osi) in time trigger defective food-seeking behavior and lethality. Further, we demonstrate that downregulation of Lipase 3, a fat body-specific lipase involved in lipid catabolism in response to starvation, rescues the reduced lipid droplet size associated with defective orsai. Finally, we show that osi-related phenotypes are rescued through the expression of its human ortholog ETFRF1/LYRm5, known to modulate the entry of ß-oxidation products into the electron transport chain; moreover, knocking down electron transport flavoproteins EtfQ0 and walrus/ETFA rescues osi-related phenotypes, further supporting this mode of action. CONCLUSIONS: These findings suggest that Osi may act in concert with the ETF complex to coordinate lipid homeostasis in the fat body in response to stage-specific demands, supporting cellular functions that in turn result in an adaptive behavioral response.

Decapentaplegic Acutely Defines the Connectivity of Central Pacemaker Neurons in Drosophila.

Rhythmic rest-activity cycles are controlled by an endogenous clock. In Drosophila, this clock resides in ∼150 neurons organized in clusters whose hierarchy changes in response to environmental conditions. The concerted activity of the circadian network is necessary for the adaptive responses to synchronizing environmental stimuli. Thus far, work was devoted to unravel the logic of the coordination of different clusters focusing on neurotransmitters and neuropeptides. We further explored communication in the adult male brain through ligands belonging to the bone morphogenetic protein (BMP) pathway. Herein we show that the lateral ventral neurons (LNvs) express the small morphogen decapentaplegic (DPP). DPP expression in the large LNvs triggered a period lengthening phenotype, the downregulation of which caused reduced rhythmicity and affected anticipation at dawn and dusk, underscoring DPP per se conveys time-of-day relevant information. Surprisingly, DPP expression in the large LNvs impaired circadian remodeling of the small LNv axonal terminals, likely through local modulation of the guanine nucleotide exchange factor Trio. These findings open the provocative possibility that the BMP pathway is recruited to strengthen/reduce the connectivity among specific clusters along the day and thus modulate the contribution of the clusters to the circadian network.SIGNIFICANCE STATEMENT The circadian clock relies on the communication between groups of so-called clock neurons to coordinate physiology and behavior to the optimal times across the day, predicting and adapting to a changing environment. The circadian network relies on neurotransmitters and neuropeptides to fine-tune connectivity among clock neurons and thus give rise to a coherent output. Herein we show that decapentaplegic, a ligand belonging to the BMP retrograde signaling pathway required for coordinated growth during development, is recruited by a group of circadian neurons in the adult brain to trigger structural remodeling of terminals on a daily basis.

The Underlying Genetics of Drosophila Circadian Behaviors.

Life is shaped by circadian clocks. This review focuses on how behavioral genetics in the fruit fly unveiled what is known today about circadian physiology. We will briefly summarize basic properties of the clock and focus on some clock-controlled behaviors to highlight how communication between central and peripheral oscillators defines their properties.

Mmp1 processing of the PDF neuropeptide regulates circadian structural plasticity of pacemaker neurons.

In the Drosophila brain, the neuropeptide PIGMENT DISPERSING FACTOR (PDF) is expressed in the small and large Lateral ventral neurons (LNvs) and regulates circadian locomotor behavior. Interestingly, PDF immunoreactivity at the dorsal terminals changes across the day as synaptic contacts do as a result of a remarkable remodeling of sLNv projections. Despite the relevance of this phenomenon to circuit plasticity and behavior, the underlying mechanisms remain poorly understood. In this work we provide evidence that PDF along with matrix metalloproteinases (Mmp1 and 2) are key in the control of circadian structural remodeling. Adult-specific downregulation of PDF levels per se hampers circadian axonal remodeling, as it does altering Mmp1 or Mmp2 levels within PDF neurons post-developmentally. However, only Mmp1 affects PDF immunoreactivity at the dorsal terminals and exerts a clear effect on overt behavior. In vitro analysis demonstrated that PDF is hydrolyzed by Mmp1, thereby suggesting that Mmp1 could directly terminate its biological activity. These data demonstrate that Mmp1 modulates PDF processing, which leads to daily structural remodeling and circadian behavior.

Acetylcholine from Visual Circuits Modulates the Activity of Arousal Neurons in Drosophila.

Drosophila melanogaster's large lateral ventral neurons (lLNvs) are part of both the circadian and sleep-arousal neuronal circuits. In the past, electrophysiological analysis revealed that lLNvs fire action potentials (APs) in bursting or tonic modes and that the proportion of neurons firing in those specific patterns varies circadianly. Here, we provide evidence that lLNvs fire in bursts both during the day and at night and that the frequency of bursting is what is modulated in a circadian fashion. Moreover, we show that lLNvs AP firing is not only under cell autonomous control, but is also modulated by the network, and in the process we develop a novel preparation to assess this. We demonstrate that lLNv bursting mode relies on a cholinergic input because application of nicotinic acetylcholine receptor antagonists impairs this firing pattern. Finally, we found that bursting of lLNvs depends on an input from visual circuits that includes the cholinergic L2 monopolar neurons from the lamina. Our work sheds light on the physiological properties of lLNvs and on a neuronal circuit that may provide visual information to these important arousal neurons. SIGNIFICANCE STATEMENT: Circadian rhythms are important for organisms to be able to anticipate daily changes in environmental conditions to adjust physiology and behavior accordingly. These rhythms depend on an endogenous mechanism that operates in dedicated neurons. In the fruit fly, the large lateral ventral neurons (lLNvs) are part of both the circadian and sleep-arousal neuronal circuits. Here, we provide new details about the firing properties of these neurons and demonstrate that they depend, not only on cell-autonomous mechanisms, but also on a specific neurotransmitter derived from visual circuits. Our work sheds light on the physiological properties of lLNvs and on a neuronal circuit that may provide visual information to these important arousal neurons.

A methyl transferase links the circadian clock to the regulation of alternative splicing.

Circadian rhythms allow organisms to time biological processes to the most appropriate phases of the day-night cycle. Post-transcriptional regulation is emerging as an important component of circadian networks, but the molecular mechanisms linking the circadian clock to the control of RNA processing are largely unknown. Here we show that PROTEIN ARGININE METHYL TRANSFERASE 5 (PRMT5), which transfers methyl groups to arginine residues present in histones and Sm spliceosomal proteins, links the circadian clock to the control of alternative splicing in plants. Mutations in PRMT5 impair several circadian rhythms in Arabidopsis thaliana and this phenotype is caused, at least in part, by a strong alteration in alternative splicing of the core-clock gene PSEUDO RESPONSE REGULATOR 9 (PRR9). Furthermore, genome-wide studies show that PRMT5 contributes to the regulation of many pre-messenger-RNA splicing events, probably by modulating 5'-splice-site recognition. PRMT5 expression shows daily and circadian oscillations, and this contributes to the mediation of the circadian regulation of expression and alternative splicing of a subset of genes. Circadian rhythms in locomotor activity are also disrupted in dart5-1, a mutant affected in the Drosophila melanogaster PRMT5 homologue, and this is associated with alterations in splicing of the core-clock gene period and several clock-associated genes. Our results demonstrate a key role for PRMT5 in the regulation of alternative splicing and indicate that the interplay between the circadian clock and the regulation of alternative splicing by PRMT5 constitutes a common mechanism that helps organisms to synchronize physiological processes with daily changes in environmental conditions.

Retrograde bone morphogenetic protein signaling shapes a key circadian pacemaker circuit.

The neuropeptide pigment-dispersing factor (PDF) synchronizes molecular oscillations within circadian pacemakers in the Drosophila brain. It is expressed in the small ventral lateral neurons (sLNvs) and large ventral lateral neurons, the former being indispensable for maintaining behavioral rhythmicity under free-running conditions. How PDF circuits develop the specific connectivity traits that endow such global behavioral control remains unknown. Here, we show that mature sLNv circuits require PDF signaling during early development, acting through its cognate receptor PDFR at postsynaptic targets. Yet, axonal defects by PDF knockdown are presynaptic and become apparent only after metamorphosis, highlighting a delayed response to a signal released early on. Presynaptic expression of constitutively active bone morphogenetic protein (BMP) receptors prevents pdfr mutants misrouting phenotype, while sLNv-restricted downregulation of BMP signaling components phenocopied pdf(01). Thus, we have uncovered a novel mechanism that provides an early "tagging" of synaptic targets that will guide circuit refinement later in development.

Organismal landscape of clock cells and circadian gene expression in Drosophila.

Background: Circadian rhythms time physiological and behavioral processes to 24-hour cycles. It is generally assumed that most cells contain self-sustained circadian clocks that drive circadian rhythms in gene expression that ultimately generating circadian rhythms in physiology. While those clocks supposedly act cell autonomously, current work suggests that in Drosophila some of them can be adjusted by the brain circadian pacemaker through neuropeptides, like the Pigment Dispersing Factor (PDF). Despite these findings and the ample knowledge of the molecular clockwork, it is still unknown how circadian gene expression in Drosophila is achieved across the body. Results: Here, we used single-cell and bulk RNAseq data to identify cells within the fly that express core-clock components. Surprisingly, we found that less than a third of the cell types in the fly express core-clock genes. Moreover, we identified Lamina wild field (Lawf) and Ponx-neuro positive (Poxn) neurons as putative new circadian neurons. In addition, we found several cell types that do not express core clock components but are highly enriched for cyclically expressed mRNAs. Strikingly, these cell types express the PDF receptor (Pdfr), suggesting that PDF drives rhythmic gene expression in many cell types in flies. Other cell types express both core circadian clock components and Pdfr, suggesting that in these cells, PDF regulates the phase of rhythmic gene expression. Conclusions: Together, our data suggest three different mechanisms generate cyclic daily gene expression in cells and tissues: canonical endogenous canonical molecular clock, PDF signaling-driven expression, or a combination of both.

Dorsal clock neurons claw their way out to control sleep in Drosophila.

The drive to sleep is strongly influenced by time of day, with temporal information conveyed through the circadian clock. In pursuit of the neural mechanisms underlying this process, in this issue of Neuron, Sun et al. identify a novel circuit that links circadian output neurons to sleep-promoting neurons within the mushroom bodies.

ENA/VASP downregulation triggers cell death by impairing axonal maintenance in hippocampal neurons.

Neurodegenerative diseases encompass a broad variety of motor and cognitive disorders that are accompanied by death of specific neuronal populations or brain regions. Cellular and molecular mechanisms underlying these complex disorders remain largely unknown. In a previous work we searched for novel Drosophila genes relevant for neurodegeneration and singled out enabled (ena), which encodes a protein involved in cytoskeleton remodeling. To extend our understanding on the mechanisms of ENA-triggered degeneration we now investigated the effect of silencing ena ortholog genes in mouse hippocampal neurons. We found that ENA/VASP downregulation led to neurite retraction and concomitant neuronal cell death through an apoptotic pathway. Remarkably, this retraction initially affected the axonal structure, showing no effect on dendrites. Reduction in ENA/VASP levels blocked the neuritogenic effect of a specific RhoA kinase (ROCK) inhibitor, thus suggesting that these proteins could participate in the Rho-signaling pathway. Altogether these observations demonstrate that ENA/VASP proteins are implicated in the establishment and maintenance of the axonal structure and that a change on their expression levels triggers neuronal degeneration.

Circadian Structural Plasticity Drives Remodeling of E Cell Output.

Behavioral outputs arise as a result of highly regulated yet flexible communication among neurons. The Drosophila circadian network includes 150 neurons that dictate the temporal organization of locomotor activity; under light-dark (LD) conditions, flies display a robust bimodal pattern. The pigment-dispersing factor (PDF)-positive small ventral lateral neurons (sLNv) have been linked to the generation of the morning activity peak (the "M cells"), whereas the Cryptochrome (CRY)-positive dorsal lateral neurons (LNds) and the PDF-negative sLNv are necessary for the evening activity peak (the "E cells") [1, 2]. While each group directly controls locomotor output pathways [3], an interplay between them along with a third dorsal cluster (the DN1ps) is necessary for the correct timing of each peak and for adjusting behavior to changes in the environment [4-7]. M cells set the phase of roughly half of the circadian neurons (including the E cells) through PDF [5, 8-10]. Here, we show the existence of synaptic input provided by the evening oscillator onto the M cells. Both structural and functional approaches revealed that E-to-M cell connectivity changes across the day, with higher excitatory input taking place before the day-to-night transition. We identified two different neurotransmitters, acetylcholine and glutamate, released by E cells that are relevant for robust circadian output. Indeed, we show that acetylcholine is responsible for the excitatory input from E cells to M cells, which show preferential responsiveness to acetylcholine during the evening. Our findings provide evidence of an excitatory feedback between circadian clusters and unveil an important plastic remodeling of the E cells' synaptic connections.

DLGS97/SAP97 is developmentally upregulated and is required for complex adult behaviors and synapse morphology and function.

The synaptic membrane-associated guanylate kinase (MAGUK) scaffolding protein family is thought to play key roles in synapse assembly and synaptic plasticity. Evidence supporting these roles in vivo is scarce, as a consequence of gene redundancy in mammals. The genome of Drosophila contains only one MAGUK gene, discs large (dlg), from which two major proteins originate: DLGA [PSD95 (postsynaptic density 95)-like] and DLGS97 [SAP97 (synapse-associated protein)-like]. These differ only by the inclusion in DLGS97 of an L27 domain, important for the formation of supramolecular assemblies. Known dlg mutations affect both forms and are lethal at larval stages attributable to tumoral overgrowth of epithelia. We generated independent null mutations for each, dlgA and dlgS97. These allowed unveiling of a shift in expression during the development of the nervous system: predominant expression of DLGA in the embryo, balanced expression of both during larval stages, and almost exclusive DLGS97 expression in the adult brain. Loss of embryonic DLGS97 does not alter the development of the nervous system. At larval stages, DLGA and DLGS97 fulfill both unique and partially redundant functions in the neuromuscular junction. Contrary to dlg and dlgA mutants, dlgS97 mutants are viable to adulthood, but they exhibit marked alterations in complex behaviors such as phototaxis, circadian activity, and courtship, whereas simpler behaviors like locomotion and odor and light perception are spared. We propose that the increased repertoire of associations of a synaptic scaffold protein given by an additional domain of protein-protein interaction underlies its ability to integrate molecular networks required for complex functions in adult synapses.

Contribution of non-circadian neurons to the temporal organization of locomotor activity.

In the fruit fly, Drosophila melanogaster, the daily cycle of rest and activity is a rhythmic behavior that relies on the activity of a small number of neurons. The small ventral lateral neurons (sLNvs) are considered key in the control of locomotor rhythmicity. Previous work from our laboratory has showed that these neurons undergo structural remodeling on their axonal projections on a daily basis. Such remodeling endows sLNvs with the possibility to make synaptic contacts with different partners at different times throughout the day, as has been previously described. By using different genetic tools to alter membrane excitability of the sLNv putative postsynaptic partners, we tested their functional role in the control of locomotor activity. We also used optical imaging to test the functionality of these contacts. We found that these different neuronal groups affect the consolidation of rhythmic activity, suggesting that non-circadian cells are part of the circuit that controls locomotor activity. Our results suggest that new neuronal groups, in addition to the well-characterized clock neurons, contribute to the operations of the circadian network that controls locomotor activity in D. melanogaster.

Basic protocols for Drosophila S2 cell line: maintenance and transfection.

Cells in culture have been increasingly employed in the dissection of intracellular processes. They are generally easier to handle than the organism of study and certainly less complex, which facilitates testing for specific functions and protein-protein interactions. This chapter will describe the extremely simple steps required to keep a healthy S2 cell culture going.

Coimmunoprecipitation on Drosophila cells in culture.

Coimmunoprecipitation (coIP) provides evidence that two or more proteins can be found in the same complex. It can be performed in vitro (employing in vitro transcribed and translated proteins, or proteins expressed in Escherichia coli) or from transfected cells, which assess whether the interaction takes place in a more functional context. This chapter includes a general description and guidelines to carry out coIP in transfected Schneider's cells.

Reporter assays.

Transcriptional feedback loops are at the core of the molecular clockworks. As single clock genes were cloned it was compelling to develop an assay that allowed simple and direct functional testing of putative activators or repressors of transcription. This chapter includes a general description and guidelines to carry out transcriptional assays in transiently transfected Schneider's cells.

A Novel Genetic Screen Identifies Modifiers of Age-Dependent Amyloid ß Toxicity in the Drosophila Brain.

The accumulation of amyloid ß peptide (Aß) in the brain of Alzheimer's disease (AD) patients begins many years before clinical onset. Such process has been proposed to be pathogenic through the toxicity of Aß soluble oligomers leading to synaptic dysfunction, phospho-tau aggregation and neuronal loss. Yet, a massive accumulation of Aß can be found in approximately 30% of aged individuals with preserved cognitive function. Therefore, within the frame of the "amyloid hypothesis", compensatory mechanisms and/or additional neurotoxic or protective factors need to be considered and investigated. Here we describe a modifier genetic screen in Drosophila designed to identify genes that modulate toxicity of Aß42 in the CNS. The expression of Aß42 led to its accumulation in the brain and a moderate impairment of negative geotaxis at 18 days post-eclosion (d.p.e) as compared with genetic or parental controls. These flies were mated with a collection of lines carrying chromosomal deletions and negative geotaxis was assessed at 5 and 18 d.p.e. Our screen is the first to take into account all of the following features, relevant to sporadic AD: (1) pan-neuronal expression of wild-type Aß42; (2) a quantifiable complex behavior; (3) Aß neurotoxicity associated with progressive accumulation of the peptide; and (4) improvement or worsening of climbing ability only evident in aged animals. One hundred and ninety-nine deficiency (Df) lines accounting for ~6300 genes were analyzed. Six lines, including the deletion of 52 Drosophila genes with human orthologs, significantly modified Aß42 neurotoxicity in 18-day-old flies. So far, we have validated CG11796 and identified CG17249 as a strong candidate (whose human orthologs are HPD and PRCC, respectively) by using RNAi or mutant hemizygous lines. PRCC encodes proline-rich protein PRCC (ppPRCC) of unknown function associated with papillary renal cell carcinoma. HPD encodes 4-hydroxyphenylpyruvate dioxygenase (HPPD), a key enzyme in tyrosine degradation whose Df causes autosomal recessive Tyrosinemia type 3, characterized by mental retardation. Interestingly, lines with a partial Df of HPD ortholog showed increased intraneuronal accumulation of Aß42 that coincided with geotaxis impairment. These previously undetected modifiers of Aß42 neurotoxicity in Drosophila warrant further study to validate their possible role and significance in the pathogenesis of sporadic AD.

Rhythmic Behavior Is Controlled by the SRm160 Splicing Factor in Drosophila melanogaster.

Circadian clocks organize the metabolism, physiology, and behavior of organisms throughout the day-night cycle by controlling daily rhythms in gene expression at the transcriptional and post-transcriptional levels. While many transcription factors underlying circadian oscillations are known, the splicing factors that modulate these rhythms remain largely unexplored. A genome-wide assessment of the alterations of gene expression in a null mutant of the alternative splicing regulator SR-related matrix protein of 160 kDa (SRm160) revealed the extent to which alternative splicing impacts on behavior-related genes. We show that SRm160 affects gene expression in pacemaker neurons of the Drosophila brain to ensure proper oscillations of the molecular clock. A reduced level of SRm160 in adult pacemaker neurons impairs circadian rhythms in locomotor behavior, and this phenotype is caused, at least in part, by a marked reduction in period (per) levels. Moreover, rhythmic accumulation of the neuropeptide PIGMENT DISPERSING FACTOR in the dorsal projections of these neurons is abolished after SRm160 depletion. The lack of rhythmicity in SRm160-downregulated flies is reversed by a fully spliced per construct, but not by an extra copy of the endogenous locus, showing that SRm160 positively regulates per levels in a splicing-dependent manner. Our findings highlight the significant effect of alternative splicing on the nervous system and particularly on brain function in an in vivo model.

Organization of Circadian Behavior Relies on Glycinergic Transmission.

The small ventral lateral neurons (sLNvs) constitute a central circadian pacemaker in the Drosophila brain. They organize daily locomotor activity, partly through the release of the neuropeptide pigment-dispersing factor (PDF), coordinating the action of the remaining clusters required for network synchronization. Despite extensive efforts, the basic principles underlying communication among circadian clusters remain obscure. We identified classical neurotransmitters released by sLNvs through disruption of specific transporters. Adult-specific RNAi-mediated downregulation of the glycine transporter or impairment of glycine synthesis in LNv neurons increased period length by nearly an hour without affecting rhythmicity of locomotor activity. Electrophysiological recordings showed that glycine reduces spiking frequency in circadian neurons. Interestingly, downregulation of glycine receptor subunits in specific sLNv targets impaired rhythmicity, revealing involvement of glycine in information processing within the network. These data identify glycinergic inhibition of specific targets as a cue that contributes to the synchronization of the circadian network.

Guidelines for Genome-Scale Analysis of Biological Rhythms.

Genome biology approaches have made enormous contributions to our understanding of biological rhythms, particularly in identifying outputs of the clock, including RNAs, proteins, and metabolites, whose abundance oscillates throughout the day. These methods hold significant promise for future discovery, particularly when combined with computational modeling. However, genome-scale experiments are costly and laborious, yielding "big data" that are conceptually and statistically difficult to analyze. There is no obvious consensus regarding design or analysis. Here we discuss the relevant technical considerations to generate reproducible, statistically sound, and broadly useful genome-scale data. Rather than suggest a set of rigid rules, we aim to codify principles by which investigators, reviewers, and readers of the primary literature can evaluate the suitability of different experimental designs for measuring different aspects of biological rhythms. We introduce CircaInSilico, a web-based application for generating synthetic genome biology data to benchmark statistical methods for studying biological rhythms. Finally, we discuss several unmet analytical needs, including applications to clinical medicine, and suggest productive avenues to address them.