Phosphatase of Regenerating Liver-1 Selectively Times Circadian Behavior in Darkness via Function in PDF Neurons and Dephosphorylation of TIMELESS.
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ABSTRACT:
SUBMITTER: Kula-Eversole E
PROVIDER: S-EPMC7855481 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Molecular circadian clocks are interconnected via neural networks. In Drosophila, PIGMENT-DISPERSING FACTOR (PDF) acts as a master network regulator with dual functions in synchronizing molecular oscillations between disparate PDF(+) and PDF(-) circadian pacemaker neurons and controlling pacemaker neuron output. Yet the mechanisms by which PDF functions are not clear. We demonstrate that genetic inhibition of protein kinase A (PKA) in PDF(-) clock neurons can phenocopy PDF mutants while activated PKA can partially rescue PDF receptor mutants. PKA subunit transcripts are also under clock control in non-PDF DN1p neurons. To address the core clock target of PDF, we rescued per in PDF neurons of arrhythmic per⁰¹ mutants. PDF neuron rescue induced high amplitude rhythms in the clock component TIMELESS (TIM) in per-less DN1p neurons. Complete loss of PDF or PKA inhibition also results in reduced TIM levels in non-PDF neurons of per⁰¹ flies. To address how PDF impacts pacemaker neuron output, we focally applied PDF to DN1p neurons and found that it acutely depolarizes and increases firing rates of DN1p neurons. Surprisingly, these effects are reduced in the presence of an adenylate cyclase inhibitor, yet persist in the presence of PKA inhibition. We have provided evidence for a signaling mechanism (PKA) and a molecular target (TIM) by which PDF resets and synchronizes clocks and demonstrates an acute direct excitatory effect of PDF on target neurons to control neuronal output. The identification of TIM as a target of PDF signaling suggests it is a multimodal integrator of cell autonomous clock, environmental light, and neural network signaling. Moreover, these data reveal a bifurcation of PKA-dependent clock effects and PKA-independent output effects. Taken together, our results provide a molecular and cellular basis for the dual functions of PDF in clock resetting and pacemaker output.
| S-EPMC3958333 | biostudies-literature
Project description:In Drosophila, neuropeptide Pigment Dispersing Factor (PDF) is expressed in small and large ventral Lateral Neurons (sLNv and lLNv), among which sLNv are critical for activity rhythms in constant darkness. Studies show that this is mediated by rhythmic accumulation and likely secretion of PDF from sLNv dorsal projections, which in turn synchronises molecular oscillations in downstream circadian neurons. Using targeted expression of a neurodegenerative protein Huntingtin in LNv, we evoke a selective loss of neuropeptide PDF and clock protein PERIOD from sLNv soma. However, PDF is not lost from sLNv dorsal projections and lLNv. These flies are behaviourally arrhythmic in constant darkness despite persistence of PDF oscillations in sLNv dorsal projections and synchronous PERIOD oscillations in downstream circadian neurons. We find that PDF oscillations in sLNv dorsal projections are not sufficient for sustenance of activity rhythms in constant darkness and this is suggestive of an additional component that is possibly dependent on sLNv molecular clock and PDF in sLNv soma. Additionally, despite loss of PERIOD in sLNv, their activity rhythms entrain to light/dark cycles indicating that sLNv molecular clocks are not necessary for entrainment. Under constant light, these flies lack PDF from both soma and dorsal projections of sLNv, and when subjected to light/dark cycles, show morning and evening anticipation and accurately phased morning and evening peaks. Thus, under light/dark cycles, PDF in sLNv is not necessary for morning anticipation.
| S-EPMC5448722 | biostudies-literature
Project description:To compare circadian gene expression within highly discrete neuronal populations, we separately purified and characterized two adjacent but distinct groups of Drosophila adult circadian neurons: the 8 small and 10 large PDF-expressing ventral lateral neurons (s-LNvs and l-LNvs, respectively). The s-LNvs are the principal circadian pacemaker cells, whereas recent evidence indicates that the l-LNvs are involved in sleep and light-mediated arousal. Although half of the l-LNv-enriched mRNA population, including core clock mRNAs, is shared between the l-LNvs and s-LNvs, the other half is l-LNv- and s-LNv-specific. The distribution of four specific mRNAs is consistent with prior characterization of the four encoded proteins, and therefore indicates successful purification of the two neuronal types. Moreover, an octopamine receptor mRNA is selectively enriched in l-LNvs, and only these neurons respond to in vitro application of octopamine. Dissection and purification of l-LNvs from flies collected at different times indicate that these neurons contain cycling clock mRNAs with higher circadian amplitudes as well as at least a 10-fold higher fraction of oscillating mRNAs than all previous analyses of head RNA. Many of these cycling l-LNv mRNAs are well expressed but do not cycle or cycle much less well elsewhere in heads. The results suggest that RNA cycling is much more prominent in circadian neurons than elsewhere in heads and may be particularly important for the functioning of these neurons.
| S-EPMC2922133 | biostudies-literature
Project description: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.
| S-EPMC4214601 | biostudies-literature
Project description:We used real-time imaging to detect cAMP levels in neurons of intact fly brains to study the mechanisms of circadian pacemaker synchronization by the neuropeptide pigment dispersing factor (PDF) in Drosophila. PDF receptor (PDF-R) is expressed by both M (sLNv) and E (LNd) pacemaker subclasses and is coupled to G(sα) in both cases. We previously reported that PDF-R in M pacemakers elevates cAMP levels by activating the ortholog of mammalian adenylate cyclase 3 (AC3) but that AC3 disruptions had no effect on E pacemaker sensitivity to PDF. Here, we show that PDF-R in E pacemakers activates a different AC isoform, AC78C, an ortholog of mammalian AC8. Knockdown of AC78C by transgenic RNAi substantially reduces, but does not completely abrogate, PDF responses in these E pacemakers. The knockdown effect is intact when restricted to mature stages, suggesting a physiological and not a development role for AC78C in E pacemakers. The AC78C phenotype is rescued by the overexpression of AC78C but not by overexpression of the rutabaga AC. AC78C overexpression does not disrupt PDF responses in these E pacemakers, and neither AC78C knockdown nor its overexpression disrupted locomotor rhythms. Finally, knockdown of 2 AKAPs, nervy and AKAP200, partially reduces LNd PDF responses. These findings begin to identify the components of E pacemaker PDF-R signalosomes and indicate that they are distinct from PDF-R signalosomes in M pacemakers: we propose they contain AC78C and at least 1 other AC.
| S-EPMC3980953 | biostudies-literature
Project description:Drosophila melanogaster flies concentrate behavioral activity around dawn and dusk. This organization of daily activity is controlled by central circadian clock neurons, including the lateral-ventral pacemaker neurons (LN(v)s) that secrete the neuropeptide PDF (pigment dispersing factor). Previous studies have demonstrated the requirement for PDF signaling to PDF receptor (PDFR)-expressing dorsal clock neurons in organizing circadian activity. Although LN(v)s also express functional PDFR, the role of these autoreceptors has remained enigmatic. Here, we show that (1) PDFR activation in LN(v)s shifts the balance of circadian activity from evening to morning, similar to behavioral responses to summer-like environmental conditions, and (2) this shift is mediated by stimulation of the G?,s-cAMP pathway and a consequent change in PDF/neurotransmitter corelease from the LN(v)s. These results suggest another mechanism for environmental control of the allocation of circadian activity and provide new general insight into the role of neuropeptide autoreceptors in behavioral control circuits.
| S-EPMC3432947 | biostudies-literature
Project description:Discrete clusters of circadian clock neurons temporally organize daily behaviors such as sleep and wake. In Drosophila, a network of just 150 neurons drives two peaks of timed activity in the morning and evening. A subset of these neurons expresses the neuropeptide pigment dispersing factor (PDF), which is important for promoting morning behavior as well as maintaining robust free-running rhythmicity in constant conditions. Yet, how PDF acts on downstream circuits to mediate rhythmic behavior is unknown. Using circuit-directed rescue of PDF receptor mutants, we show that PDF targeting of just approximately 30 non-PDF evening circadian neurons is sufficient to drive morning behavior. This function is not accompanied by large changes in core molecular oscillators in light-dark, indicating that PDF RECEPTOR likely regulates the output of these cells under these conditions. We find that PDF also acts on this focused set of non-PDF neurons to regulate both evening activity phase and period length, consistent with modest resetting effects on core oscillators. PDF likely acts on more distributed pacemaker neuron targets, including the PDF neurons themselves, to regulate rhythmic strength. Here we reveal defining features of the circuit-diagram for PDF peptide function in circadian behavior, revealing the direct neuronal targets of PDF as well as its behavioral functions at those sites. These studies define a key direct output circuit sufficient for multiple PDF dependent behaviors.
| S-EPMC2702683 | biostudies-literature
Project description:Daily behaviors in animals are determined by the interplay between internal timing signals from circadian clocks and environmental stimuli such as light. How these signals are integrated to produce timely and adaptive behavior is unclear. The fruit fly Drosophila exhibits clock-driven activity increases that anticipate dawn and dusk and free-running rhythms under constant conditions. Flies also respond to the onset of light and dark with acute increases in activity.Mutants of a novel ion channel, narrow abdomen (na), lack a robust increase in activity in response to light and show reduced anticipatory behavior and free-running rhythms, providing a genetic link between photic responses and circadian clock function. We used tissue-specific rescue of na to demonstrate a role for approximately 16-20 circadian pacemaker neurons, a subset of the posterior dorsal neurons 1 (DN1(p)s), in mediating the acute response to the onset of light as well as morning anticipatory behavior. Circadian pacemaker neurons expressing the neuropeptide PIGMENT-DISPERSING FACTOR (PDF) are especially important for morning anticipation and free-running rhythms and send projections to the DN1(p)s. We also demonstrate that DN1(p)Pdfr expression is sufficient to rescue, at least partially, Pdfr morning anticipation defects as well as defects in free-running rhythms, including those in DN1 molecular clocks. Additionally, these DN1 clocks in wild-type flies are more strongly reset to timing changes in PDF clocks than other pacemaker neurons, suggesting that they are direct targets.Taking these results together, we demonstrate that the DN1(p)s lie at the nexus of PDF and photic signaling to produce appropriate daily behavior.
| S-EPMC2864127 | biostudies-literature
Project description:The timing of behavior under natural light-dark conditions is a function of circadian clocks and photic input pathways. Yet a mechanistic understanding of how these pathways collaborate in animals is lacking. Here we demonstrate in Drosophila that the Phosphatase of Regenerating Liver-1 (PRL-1) sets period length and behavioral phase gated by photic signals. PRL-1 knockdown in PDF clock neurons dramatically lengthens circadian period. PRL-1 mutants exhibit allele-specific interactions with the light- and clock-regulated gene timeless (tim). Moreover, we show that PRL-1 promotes TIM accumulation and dephosphorylation. Interestingly, the PRL-1 mutant period lengthening is suppressed in constant light and PRL-1 mutants display a delayed phase under short, but not long, photoperiod conditions. Thus, our studies reveal that PRL-1 dependent dephosphorylation of TIM is a core mechanism of the clock that sets period length and phase in darkness, enabling the behavioral adjustment to changing day-night cycles.
2020-11-11 | PXD020986 | Pride
Project description:The neuropeptide PDF is crucial for Drosophila circadian behavior: it keeps circadian neurons synchronized. Here, we identify GW182 as a key regulator of PDF signaling. Indeed, GW182 downregulation results in phenotypes similar to those of Pdf and Pdf-receptor (Pdfr) mutants. gw182 genetically interacts with Pdfr and cAMP signaling, which is essential for PDFR function. GW182 mediates miRNA-dependent gene silencing through its interaction with AGO1. Consistently, GW182's AGO1 interaction domain is required for GW182's circadian function. Moreover, our results indicate that GW182 modulates PDFR signaling by silencing the expression of the cAMP phosphodiesterase DUNCE. Importantly, this repression is under photic control, and GW182 activity level--which is limiting in circadian neurons--influences the responses of the circadian neural network to light. We propose that GW182's gene silencing activity functions as a rheostat for PDFR signaling and thus profoundly impacts the circadian neural network and its response to environmental inputs.
| S-EPMC3629553 | biostudies-literature