PDF and cAMP enhance PER stability in Drosophila clock neurons

Abstract

The neuropeptide PDF is important for Drosophila circadian rhythms: pdf(01) (pdf-null) animals are mostly arrhythmic or short period in constant darkness and have an advanced activity peak in light-dark conditions. PDF contributes to the amplitude, synchrony, as well as the pace of circadian rhythms within clock neurons. PDF is known to increase cAMP levels in PDR receptor (PDFR)-containing neurons. However, there is no known connection of PDF or of cAMP with the Drosophila molecular clockworks. We discovered that the mutant period gene per(S) ameliorates the phenotypes of pdf-null flies. The period protein (PER) is a well-studied repressor of clock gene transcription, and the per(S) protein (PERS) has a markedly short half-life. The result therefore suggests that the PDF-mediated increase in cAMP might lengthen circadian period by directly enhancing PER stability. Indeed, increasing cAMP levels and cAMP-mediated protein kinase A (PKA) activity stabilizes PER, in S2 tissue culture cells and in fly circadian neurons. Adding PDF to fly brains in vitro has a similar effect. Consistent with these relationships, a light pulse causes more prominent PER degradation in pdf(01) circadian neurons than in wild-type neurons. The results indicate that PDF contributes to clock neuron synchrony by increasing cAMP and PKA, which enhance PER stability and decrease clock speed in intrinsically fast-paced PDFR-containing clock neurons. We further suggest that the more rapid degradation of PERS bypasses PKA regulation and makes the pace of clock neurons more uniform, allowing them to avoid much of the asynchrony caused by the absence of PDF.

Keywords: PDF neurons; PDF signaling; molecular clock regulation; synchronization.

Fig. 1.

per^S ameliorates the loss of PDF. Flies were entrained in 12:12-h light–dark (LD) cycles at 25 °C for at least 3 d, and then moved to constant-dark conditions (DD) for 6 d. The data were collected and double plotted on an actogram. Actograms are presenting the locomotive activity of following genotypes: (A) canton-S (cs); (B) per^S; (C) rhythmic flies of pdf⁰¹; (D) rhythmic flies of per^S;;pdf⁰¹. The mean of the period for the first 5 d in DD, the total number of animals, and the percentages of rhythmic animals are shown in Table 1. The actograms of several single flies are shown in Fig. S1.

Fig. 2.

Oscillation of PER in the different clock neurons of pdf⁰¹ and per^S;;pdf⁰¹ flies in DD. Pdf⁰¹ and per^S;;pdf⁰¹ flies were entrained in 12:12 light–dark (LD) cycles at 25 °C for 3 d, and then moved to DD. Relative PER levels in DN1s, LNds, and LNvs from the brains at CT10 and CT22 were compared. Because the periods of these flies did not maintain a period of 24 h in DD (Table 1), their periods were divided by 24 to designate a specific one “hour unit.” CT10 and CT22 are the 10th and 22nd “hour units,” respectively. DD1, 5, and 6 indicate the first, fifth, and sixth period cycle in DD, respectively. Because the period of a per^S;;pdf⁰¹ fly was about 4.5 h shorter than a pdf⁰¹ fly, the time length of six cycles in a per^S;;pdf⁰¹ fly was roughly equivalent to the length of five cycles in a pdf⁰¹ fly. The histograms represent the mean relative PER intensity in the indicated neurons in pdf⁰¹ flies (A) and per^S;;pdf⁰¹ flies (B). The number of hemispheres is indicated by “n.” Error bars represent ±SEM. The triple asterisk (***) represents P < 0.001, the double asterisk (**) represents P < 0.01, and “n.s.” represents no statistical significance as determined by Student t test. The oscillations of PER in the clock neurons of WT and per^S flies are shown in Fig. S2.

Fig. 3.

The up-regulation of cAMP in S2 cells inhibits PER degradation. PER degradation rate was assayed in vitro using Drosophila S2 cells. The expression of per under the control of a heat shock promoter was induced using a 37 °C heat shock for 30 min. (A) The effect of up-regulation of cAMP on PER stability was assayed by adding cycloheximide (CHX) with or without forskolin (FSK) and Sp-cAMPS (Sp) 2 h post induction. (B) The effect of PKA on the stability of PER was examined by adding a PKA inhibitor PKI to the cells to inhibit the activity of PKA after FSK induction. Total protein was extracted at different time points after each treatment. PER was detected using an anti-PER. β-Actin was used as loading control. The histogram below the blots represents the mean relative intensity of the blots from three independent experiments. Error bars represent ±SEM. The triple asterisk (***) represents P < 0.001, and “n.s.” represents no statistical significance as determined by one-way ANOVA test.

Fig. 4.

Activation of PKA inhibits PER degradation in fly brains. Brains of light-insensitive cry⁰¹ flies were dissected and immunostained at ZT22 and ZT4 to show PER degradation during that period. As controls, fly heads collected at ZT22 and ZT4 were fixed and dissected. Another group of heads were collected and dissected at ZT22 in adult hemolymph-like media (AHL) with drugs including PKA activator Sp-cAMPS and PKA inhibitor PKI as indicated in the figure. Brains were incubated in the AHL, with or without drugs, until ZT4 before fixed. (A) PER staining (magenta) in PDF cells (green). (Scale bar: 20 µm.) (B) Quantification of PER signal in PDF cells. All results were normalized to the brain that had the strongest signal at ZT22. The number of hemispheres tested is indicated by “n.” The bars in the histogram indicate the mean relative PER intensity. Each experiment was performed two to four times, and the number of hemispheres quantified is indicated below each bar. Error bars represent ±SEM. The letters above each bar represent the statistically significant groups as determined by one-way ANOVA, Turkey post hoc test.

Fig. 5.

Degradation of PERS cannot be inhibited by PKA activation. Brains of per^S;;cry⁰¹ flies were immunostained using anti-PER antibody. PER signal in PDF neurons was quantified as described in Fig. 4. (A) PER staining (magenta) in PDF cells (green). (Scale bar: 20 µm.) (B) Quantification of PER signal in PDF cells. The histogram shows the mean relative PER intensity and the number of hemispheres quantified. Error bars represent ±SEM. The letters above each bar represent the statistically significant groups as determined by one-way ANOVA, Turkey post hoc test.

Fig. 6.

In vitro addition of PDF stabilizes PER in fly brains. Same experiment was performed as in Fig. 4, except that 10 µM PDF peptide instead of Sp-cAMPS was added to the AHL. Brains were dissected at ZT22 in AHL with peptide and drug as indicated in the figure. The brains were incubated for 6 h until ZT4 before and fixed. (A) PER staining (magenta) in PDF cells (green). (Scale bar: 20 µm.) (B) PER signal in PDF cells was quantified. Data were analyzed as in Fig. 4. The bars in the histogram indicate the mean relative PER intensity. The number of brain hemispheres tested in two independent experiments is indicated by “n.” Error bars represent ±SEM. The triple asterisk (***) represents P < 0.001, and “n.s.” represents no statistical significance as determined by one-way ANOVA, Turkey post hoc test.

Fig. 7.

PDF protects PER from light-induced degradation at ZT18. A 10-min light pulse was given to flies at ZT18. Fly brains were dissected and stained at ZT19 and compared with flies from ZT19 with no light pulse. (A) In cs flies, PER staining (magenta) in PDF cells (green, PDF neurons immunostained with anti-PDF antibody) with or without the light pulse at ZT18. In UAS-mCD8-GFP; pdf-GAL4; pdf⁰¹ flies, PER staining (magenta) in PDF cells (green, PDF neurons were immunostained with anti-GFP) with or without a light pulse at ZT18. (Scale bar: 20 µm.) (B) PER signal in PDF cells from both genotypes with or without a light pulse was quantified. All results were normalized to the cs brain with the highest signal intensity at ZT19. The experiment was performed twice. The bars in the histogram indicate the mean relative PER intensity. The number of brain hemisphere tested is indicated by “n.” Error bars represent ±SEM. The triple asterisk (***) represents P < 0.001, and “n.s.” represents no statistical significance as determined by Student t test.