Mutants which carry mutations in genes encoding mitochondrial ligases MUL1 and PARKIN are convenient Drosophila models of Parkinson’s disease (PD). In several studies it has been shown that in Parkinson’s disease sleep disturbance occurs, which may be the result of a disturbed circadian clock. We found that the ROS level was higher, while the anti-oxidant enzyme SOD1 level was lower in mul1A6 and park1 mutants than in the white mutant used as a control. Moreover, mutations of both ligases affected circadian rhythms and the clock. The expression of clock genes per, tim and clock and the level of PER protein were changed in the mutants. Moreover, expression of ATG5, an autophagy protein also involved in circadian rhythm regulation, was decreased in the brain and in PDF-immunoreactive large ventral lateral clock neurons. The observed changes in the molecular clock resulted in a longer period of locomotor activity rhythm, increased total activity and shorter sleep at night. Finally, the lack of both ligases led to decreased longevity and climbing ability of the flies. All of the changes observed in the brains of these Drosophila models of PD, in which mitochondrial ligases MUL1 and PARKIN do not function, may explain the mechanisms of some neurological and behavioural symptoms of PD.

Light is one of the most important factors regulating rhythmical behavior of Drosophila melanogaster. It is received by different photoreceptors and entrains the circadian clock, which controls sleep. The retina is known to be essential for light perception, as it is composed of specialized light-sensitive cells which transmit signal to deeper parts of the brain. In this study we examined the role of specific photoreceptor types and peripheral oscillators located in these cells in the regulation of sleep pattern. We showed that sleep is controlled by the visual system in a very complex way. Photoreceptors expressing Rh1, Rh3 are involved in night-time sleep regulation, while cells expressing Rh5 and Rh6 affect sleep both during the day and night. Moreover, Hofbauer-Buchner (HB) eyelets which can directly contact with s-LN_vs and l-LN_vs play a wake-promoting function during the day. In addition, we showed that L2 interneurons, which receive signal from R1-6, form direct synaptic contacts with l-LN_vs, which provides new light input to the clock network.

Drosophila Cryptochrome (CRY) is a blue light sensitive protein with a key role in circadian photoreception. A main feature of CRY is that light promotes an interaction with the circadian protein TIMELESS (TIM) resulting in their ubiquitination and degradation, a mechanism that contributes to the synchronization of the circadian clock to the environment. Moreover, CRY participates in non-circadian functions such as magnetoreception, modulation of neuronal firing, phototransduction and regulation of synaptic plasticity. In the present study we used co-immunoprecipitation, yeast two hybrid and in situ proximity ligation assay to show that CRY can physically associate with the presynaptic protein Bruchpilot (BRP) and that CRY-BRP complexes are located mainly in the visual system. Additionally, we present evidence that light-activated CRY may decrease BRP levels in photoreceptor termini in the distal lamina, probably targeting BRP for degradation.

The daily expression of genes and the changes in gene expression after silencing the heme oxygenase (ho) gene were examined in the retina of Drosophila using microarray and SybrGreen qPCR (quantitative polymerase chain reaction) methods. The HO decrease in the morning upregulated 83 genes and downregulated 57 genes. At night, 80 genes were upregulated and 22 were downregulated. The top 20 genes downregulated after ho silencing in the morning modulate phototransduction, immune responses, autophagy, phagocytosis, apoptosis, the carbon monoxide (CO) response, the oxidative stress/UV response, and translation. In turn, the genes that upregulated at night were involved in translation—the response to oxidative stress, DNA damage, and phototransduction. Among the top 20 genes downregulated at night were genes involved in phototransduction, immune responses, and autophagy. For some genes, a low level of HO had an opposite effect in the morning compared to those at night. Silencing ho also changed the expression of circadian clock genes, while the HO decrease during the night enhanced the expression of immune system genes. The results showed that the cyclic expression of HO is important for controlling several processes in the retina, including neuroprotection and those involved in the innate immune system.

In Drosophila melanogaster, the pacemaker located in the brain plays the main role in maintaining circadian rhythms; however, peripheral oscillators including glial cells, are also crucial components of the circadian network. In the present study, we investigated an impact of oscillators located in astrocyte-like glia, the chiasm giant glia of the optic lobe, epithelial and subperineurial glia on sleep of Drosophila males. We described that oscillators located in astrocyte-like glia and chiasm giant glia are necessary to maintain daily changes in clock neurons arborizations, while those located in epithelial glia regulate amplitude of these changes. Finally, we showed that communication between glia and neurons through tripartite synapses formed by epithelial glia and, in effect, neurotransmission regulation plays important role in wake-promoting during the day. SIGNIFICANCE STATEMENT Circadian clock or pacemaker regulates many aspects of animals’ physiology and behavior. The pacemaker is located in the brain and is composed of neurons. However, there are also additional oscillators, called peripheral clocks, which synchronize the main clock. Despite the critical role of glia in the clock machinery, little is known which type of glia houses peripheral oscillators and how they affect neuronal clocks. This study using Drosophila shows that oscillators in specific glia types maintain awakeness during the day by regulating the daily plasticity of clock neurons.

Autophagy is a self-degradative process which plays a role in removing misfolded or aggregated proteins, clearing damaged organelles, but also in changes of cell membrane size and shape. The aim of this phenomenon is to deliver cytoplasmic cargo to the lysosome through the intermediary of a double membrane-bound vesicle (autophagosome), that fuses with a lysosome to form autolysosome, where cargo is degraded by proteases. Products of degradation are transported back to the cytoplasm, where they can be re-used. In the present study we showed that autophagy is important for proper functioning of the glia and that it is involved in the regulation of circadian structural changes in processes of the pacemaker neurons. This effect is mainly observed in astrocyte-like glia, which play a role of peripheral circadian oscillators in the Drosophila brain.