TOR signaling pathway and autophagy are involved in the regulation of circadian rhythms in behavior and plasticity of L2 interneurons in the brain of Drosophila melanogaster

Abstract

Drosophila melanogaster is a common model used to study circadian rhythms in behavior and circadian clocks. However, numerous circadian rhythms have also been detected in non-clock neurons, especially in the first optic neuropil (lamina) of the fly's visual system. Such rhythms have been observed in the number of synapses and in the structure of interneurons, which exhibit changes in size and shape in a circadian manner. Although the patterns of these changes are known, the mechanism remains unclear. In the present study, we investigated the role of the TOR signaling pathway and autophagy in regulating circadian rhythms based on the behavior and structural plasticity of the lamina L2 monopolar cell dendritic trees. In addition, we examined the cyclic expression of the TOR signaling pathway (Tor, Pi3K class 1, Akt1) and autophagy (Atg5 and Atg7) genes in the fly's brain. We observed that Tor, Atg5 and Atg7 exhibit rhythmic expressions in the brain of wild-type flies in day/night conditions (LD 12:12) that are abolished in per01 clock mutants. The silencing of Tor in per expressing cells shortens a period of the locomotor activity rhythm of flies. In addition, silencing of the Tor and Atg5 genes in L2 cells disrupts the circadian plasticity of the L2 cell dendritic trees measured in the distal lamina. In turn, silencing of the Atg7 gene in L2 cells changes the pattern of this rhythm. Our results indicate that the TOR signaling pathway and autophagy are involved in the regulation of circadian rhythms in the behavior and plasticity of neurons in the brain of adult flies.

Fig 1. Transgenic flies (21D-GAL4>UAS-mCD8-GFP) with targeted GFP expression to L2 cell membranes.

(A)—The L2 cell bodies are located in the lamina cortex (*), axons with dendrites in the lamina synaptic neuropil (La) and terminals of axons in the second optic neuropil (Me—medulla); R–retina, arrows show the L2 cell terminals in the medulla. (B)–Cross-section of the lamina with GFP-labeled L2 cells with dendritic trees. (C)–A deconvolved image of L2 dendritic trees for which the perimeter was measured during analyses.

Fig 2. The relative level of TOR signaling pathway and autophagy genes RNA in the fly’s brain.

A—The Tor gene RNA cycles only in the brains of Canton S in LD 12:12, reaching the highest level 4 h before the end of the night (ZT20) and the lowest 4 h after the beginning of the day (ZT4) (mean RQ +/- SE) [Kruskal-Wallis Test: H (5, N = 25) = 15,10543 p =, 0099; post hoc multiple comparison test: * < 0.05]. B—The relative level of Atg5 RNA in the brain of Canton S male flies, held in LD 12:12 or in DD and in per⁰¹ mutants in LD 12:12 (mean RQ +/- SE). The Atg5 RNA cycles in the brains of Canton S in LD 12:12, reaching the highest level 4 h after the beginning of the day (ZT4) and 4 h before the end of the night (ZT20) [Kruskal-Wallis test: H (5, N = 35) = 23,34957 p =, 0003, post hoc multiple comparison test: * < 0.05]. C—The relative level of Atg7 RNA in the brain of Canton S male flies, held in LD 12:12 or in DD and in per⁰¹ mutants in LD 12:12 (mean RQ +/- SE). The Atg7 RNA cycles in the brain of Canton S in LD 12:12. The highest level was detected 4 h before the end of the night (ZT20) and at the beginning of the day (ZT1) [Kruskal-Wallis test: H (5, N = 31) = 23,07912 p =, 0003, post hoc multiple comparison test: * < 0.05; ** < 0.01].

Fig 3. Periods of the locomotor activity rhythm of flies after silencing the expressions of studied genes.

Tor, Tsc1, Rheb, Atg5 and Atg7 genes expression was silenced in per-positive cells. Period of locomotor activity rhythm was measured in DD conditions (mean +/- SE). Representative actograms were presented. As a control, the progeny of per-Gal4 and UAS-Val10-GFP crossing was used. After silencing the Tor gene, the period of locomotor activity rhythm was significantly shorter than in the control [U Mann-Whitney Test, *** for p≤0,001]. Silencing the Tsc1 gene caused a slight lengthening in the period of the rhythm.

Fig 4. Sleep and activity of flies with silenced expression of TOR signaling pathway and autophagy genes.

(A)—The total activity of flies recorded on the second day of experiment (mean +/- SD). Silencing the Tsc1 gene in per-positive cells increased the total activity of the flies [U Mann-Whitney Test, * for p≤0,05]. (B)—Total sleep of flies in the dark phase (mean +/- SD). The flies with silenced expressions of Tor or Atg7 gene had lengthened sleep durations in the dark [U Mann-Whitney Test, ** for p≤0,01, * for p≤0,5]. (C)–The sleep metrics of flies with silenced expression of the Tor gene in per-positive cells (x axis shows the time in hours). These flies were less active than control flies during the morning peak of activity but were more active during the evening peaks. (D)—The sleep metrics of flies with silenced expression of the Tsc1 gene in per-positive cells. The experimental flies were more active than control flies during the entire recorded period, regardless of the time of day. (E)—The sleep metrics of flies with silenced expression of the Rheb gene in per-positive cells. Experimental flies were less active during morning peaks of activity in comparison to control flies. (F)—The sleep metrics of flies with silenced expression of the Atg7 gene in per-positive cells. (G)—The sleep metrics of flies with silenced expression of the Atg5 gene in per-positive cells.

Fig 5. The perimeter of L2 cell dendritic trees after silencing Tor, Atg5 and Atg7 genes.

After silencing the Tor (A) or Atg5 (B) under control of the 21D-promoter, the daily rhythm of the L2 cell dendritic tree size was abolished (mean +/- SD) [Tor: ANOVA, p>0.05; Atg5: Kruskal-Wallis Test: H (3, N = 401) = 7,722824 p =, 0521], while in case of Atg7 (C), the pattern of the daily rhythm of the L2 dendritic trees was changed. The dendritic trees were largest at the beginning of the day, and their size decreased during the day and at night [Kruskal-Wallis Test: H (3, N = 412) = 40.63845 p = 0.0000; multiple comparison test, ***—p≤0.01]. The differences between time points in control flies are indicated with “a” and “b” letters. The dendritic trees are largest at the beginning of the night, in ZT13—a [Kruskal-Wallis test: H (3, N = 423) = 23,31382 p =, 0000; multiple comparison test, the difference between ZT13 and ZT4—p≤0.01, differences among ZT13 and ZT1 and ZT13 and ZT16 p≤0.001].