Autophagy as a new player in the regulation of clock neurons physiology of Drosophila melanogaster.

Axonal terminals of the small ventral lateral neurons (sLNvs), the circadian clock neurons of Drosophila, show daily changes in their arborization complexity, with many branches in the morning and their shrinkage during the night. This complex phenomenon is precisely regulated by several mechanisms. In the present study we describe that one of them is autophagy, a self-degradative process, also involved in changes of cell membrane size and shape. Our results showed that autophagosome formation and processing in PDF-expressing neurons (both sLNv and lLNv) are rhythmic and they have different patterns in the cell bodies and terminals. These rhythmic changes in the autophagy activity seem to be important for neuronal plasticity. We found that autophagosome cargos are different during the day and night, and more proteins involved in membrane remodeling are present in autophagosomes in the morning. In addition, we described for the first time that Atg8-positive vesicles are also present outside the sLNv terminals, which suggests that secretory autophagy might be involved in regulating the clock signaling network. Our data indicate that rhythmic autophagy in clock neurons affect the pacemaker function, through remodeling of terminal membrane and secretion of specific proteins from sLNvs.

A PDZ scaffolding/CaM-mediated pathway in Cryptochrome signaling.

Cryptochromes are cardinal constituents of the circadian clock, which orchestrates daily physiological rhythms in living organisms. A growing body of evidence points to their participation in pathways that have not traditionally been associated with circadian clock regulation, implying that cryptochromes may be subject to modulation by multiple signaling mechanisms. In this study, we demonstrate that human CRY2 (hCRY2) forms a complex with the large, modular scaffolding protein known as Multi-PDZ Domain Protein 1 (MUPP1). This interaction is facilitated by the calcium-binding protein Calmodulin (CaM) in a calcium-dependent manner. Our findings suggest a novel cooperative mechanism for the regulation of mammalian cryptochromes, mediated by calcium ions (Ca2+ ) and CaM. We propose that this Ca2+ /CaM-mediated signaling pathway may be an evolutionarily conserved mechanism that has been maintained from Drosophila to mammals, most likely in relation to its potential role in the broader context of cryptochrome function and regulation. Further, the understanding of cryptochrome interactions with other proteins and signaling pathways could lead to a better definition of its role within the intricate network of molecular interactions that govern circadian rhythms.

Changes in heme oxygenase level during development affect the adult life of Drosophila melanogaster.

Heme oxygenase (HO) has been shown to control various cellular processes in both mammals and Drosophila melanogaster. Here, we investigated how changes in HO levels in neurons and glial cells during development affect adult flies, by using the TARGET Drosophila system to manipulate the expression of the ho gene. The obtained data showed differences in adult survival, maximum lifespan, climbing, locomotor activity, and sleep, which depended on the level of HO (after ho up-regulation or downregulation), the timing of expression (chronic or at specific developmental stages), cell types (neurons or glia), sex (males or females), and age of flies. In addition to ho, the effects of changing the mRNA level of the Drosophila CNC factor gene (NRF2 homolog in mammals and master regulator of HO), were also examined to compare with those observed after changing ho expression. We showed that HO levels in neurons and glia must be maintained at an appropriate physiological level during development to ensure the well-being of adults. We also found that the downregulation of ho in either neurons or glia in the brain is compensated by ho expressed in the retina.

The dual role of heme oxygenase in regulating apoptosis in the nervous system of Drosophila melanogaster.

Accumulating evidence from mammalian studies suggests the dual-faced character of heme oxygenase (HO) in oxidative stress-dependent neurodegeneration. The present study aimed to investigate both neuroprotective and neurotoxic effects of heme oxygenase after the ho gene chronic overexpression or silencing in neurons of Drosophila melanogaster. Our results showed early deaths and behavioral defects after pan-neuronal ho overexpression, while survival and climbing in a strain with pan-neuronal ho silencing were similar over time with its parental controls. We also found that HO can be pro-apoptotic or anti-apoptotic under different conditions. In young (7-day-old) flies, both the cell death activator gene (hid) expression and the initiator caspase Dronc activity increased in heads of flies when ho expression was changed. In addition, various expression levels of ho produced cell-specific degeneration. Dopaminergic (DA) neurons and retina photoreceptors are particularly vulnerable to changes in ho expression. In older (30-day-old) flies, we did not detect any further increase in hid expression or enhanced degeneration, however, we still observed high activity of the initiator caspase. In addition, we used curcumin to further show the involvement of neuronal HO in the regulation of apoptosis. Under normal conditions, curcumin induced both the expression of ho and hid, which was reversed after exposure to high-temperature stress and when supplemented in flies with ho silencing. These results indicate that neuronal HO regulates apoptosis and this process depends on ho expression level, age of flies, and cell type.

Light exposure during development affects physiology of adults in Drosophila melanogaster.

Light is one of most important factors synchronizing organisms to day/night cycles in the environment. In Drosophila it is received through compound eyes, Hofbauer-Buchner eyelet, ocelli, using phospholipase C-dependent phototransduction and by deep brain photoreceptors, like Cryptochrome. Even a single light pulse during early life induces larval-time memory, which synchronizes the circadian clock and maintains daily rhythms in adult flies. In this study we investigated several processes in adult flies after maintaining their embryos, larvae and pupae in constant darkness (DD) until eclosion. We found that the lack of external light during development affects sleep time, by reduction of night sleep, and in effect shift to the daytime. However, disruption of internal CRY- dependent photoreception annuls this effect. We also observed changes in the expression of genes encoding neurotransmitters and their receptors between flies kept in different light regime. In addition, the lack of light during development results in decreasing size of mushroom bodies, involved in sleep regulation. Taking together, our results show that presence of light during early life plays a key role in brain development and affects adult behavior.

The Role of Glia Clocks in the Regulation of Sleep in Drosophila melanogaster.

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 STATEMENTCircadian 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.

Antimicrobial Properties of a Peptide Derived from the Male Fertility Factor kl2 Protein of Drosophila melanogaster.

Antimicrobial peptides (AMPs) are important components of innate immunity. Here, we report the antimicrobial properties of a peptide derived from the Male fertility factor kl2 (MFF-kl2) protein of Drosophila melanogaster, which was identified as a functional analog of the mammalian antibacterial chemerin-p4 peptide. The antimicrobial activity of multifunctional chemerin is mainly associated with a domain localized in the middle of the chemerin sequence, Val66-Pro85 peptide (chemerin-p4). Using bioinformatic tools, we found homologs of the chemerin-p4 peptide in the proteome of D. melanogaster. One of them is MFF-p1, which is a part of the MFF kl2 protein, encoded by the gene male fertility factor kl2 (kl-2) located on the long arm of the Y chromosome. The second detected peptide (Z-p1) is a part of the Zizimin protein belonging to DOCK family, which is involved in cellular signaling processes. After testing the antimicrobial properties of both peptides, we found that only MFF-p1 possesses these properties. Here, we demonstrate its antimicrobial potential both in vitro and in vivo after infecting D. melanogaster with bacteria. MFF-p1 strongly inhibits the viable counts of E. coli and B. subtilis after 2 h of treatment and disrupts bacterial cells. The expression of kl-2 is regulated by exposure to bacteria and by the circadian clock.

Glia-Neurons Cross-Talk Regulated Through Autophagy.

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.

Pigment Dispersing Factor Is a Circadian Clock Output and Regulates Photoperiodic Response in the Linden Bug, Pyrrhocoris apterus.

Daily and annually cycling conditions manifested on the Earth have forced organisms to develop time-measuring devices. Circadian clocks are responsible for adjusting physiology to the daily cycles in the environment, while the anticipation of seasonal changes is governed by the photoperiodic clock. Circadian clocks are cell-autonomous and depend on the transcriptional/translational feedback loops of the conserved clock genes. The synchronization among clock centers in the brain is achieved by the modulatory function of the clock-dependent neuropeptides. In insects, the most prominent clock neuropeptide is Pigment Dispersing Factor (PDF). Photoperiodic clock measures and computes the day and/or night length and adjusts physiology accordingly to the upcoming season. The exact mechanism of the photoperiodic clock and its direct signaling molecules are unknown but, in many insects, circadian clock genes are involved in the seasonal responses. While in Drosophila, PDF signaling participates both in the circadian clock output and in diapause regulation, the weak photoperiodic response curve of D. melanogaster is a major limitation in revealing the full role of PDF in the photoperiodic clock. Here we provide the first description of PDF in the linden bug, Pyrrhocoris apterus, an organism with a robust photoperiodic response. We characterize in detail the circadian and photoperiodic phenotype of several CRISPR/Cas9-generated pdf mutants, including three null mutants and two mutants with modified PDF. Our results show that PDF acts downstream of CRY and plays a key role as a circadian clock output. Surprisingly, in contrast to the diurnal activity of wild-type bugs, pdf null mutants show predominantly nocturnal activity, which is caused by the clock-independent direct response to the light/dark switch. Moreover, we show that together with CRY, PDF is involved in the photoperiod-dependent diapause induction, however, its lack does not disrupt the photoperiodic response completely, suggesting the presence of additional clock-regulated factors. Taken together our data provide new insight into the role of PDF in the insect's circadian and photoperiodic systems.

Loss of Timeless Underlies an Evolutionary Transition within the Circadian Clock.

Most organisms possess time-keeping devices called circadian clocks. At the molecular level, circadian clocks consist of transcription-translation feedback loops (TTFLs). Although some components of the negative TTFL are conserved across the animals, important differences exist between typical models, such as mouse and the fruit fly. In Drosophila, the key components are PERIOD (PER) and TIMELESS (TIM-d) proteins, whereas the mammalian clock relies on PER and CRYPTOCHROME (CRY-m). Importantly, how the clock has maintained functionality during evolutionary transitions between different states remains elusive. Therefore, we systematically described the circadian clock gene setup in major bilaterian lineages and identified marked lineage-specific differences in their clock constitution. Then we performed a thorough functional analysis of the linden bug Pyrrhocoris apterus, an insect species comprising features characteristic of both the Drosophila and the mammalian clocks. Unexpectedly, the knockout of timeless-d, a gene essential for the clock ticking in Drosophila, did not compromise rhythmicity in P. apterus, it only accelerated its pace. Furthermore, silencing timeless-m, the ancestral timeless type ubiquitously present across animals, resulted in a mild gradual loss of rhythmicity, supporting its possible participation in the linden bug clock, which is consistent with timeless-m role suggested by research on mammalian models. The dispensability of timeless-d in P. apterus allows drawing a scenario in which the clock has remained functional at each step of transition from an ancestral state to the TIM-d-independent PER + CRY-m system operating in extant vertebrates, including humans.

Regulation of Heme Oxygenase and Its Cross-Talks with Apoptosis and Autophagy under Different Conditions in Drosophila.

Heme oxygenase (HO) is one of the cytoprotective enzymes that can mitigate the effects of oxidative stress. Here, we found that the ho mRNA level oscillates in the brain of Drosophila melanogaster with two minima at the beginning of the day and night. This rhythm was partly masked by light as its pattern changed in constant darkness (DD). It followed a similar trend in the clock mutant per01 under light/dark regime (LD12:12); however, differences between time points were not statistically significant. In older flies (20 days old), the rhythm was vanished; however, 15 days of curcumin feeding restored this rhythm with an elevated ho mRNA level at all time points studied. In addition, flies exposed to paraquat had higher ho expression in the brain, but only at a specific time of the day which can be a protective response of the brain against stress. These findings suggest that the expression of ho in the fly's brain is regulated by the circadian clock, light, age, exposure to stress, and the presence of exogenous antioxidants. We also found that HO cross-talks with apoptosis and autophagy under different conditions. Induction of neuronal ho was accompanied by increased transcription of apoptosis and autophagy-related genes. However, this trend changed after exposure to curcumin and paraquat. Our results suggest that HO is involved in the control of apoptotic and autophagic key processes protecting the brain against oxidative damage.

Better Sleep at Night: How Light Influences Sleep in Drosophila.

Sleep-like states have been described in Drosophila and the mechanisms and factors that generate and define sleep-wake profiles in this model organism are being thoroughly investigated. Sleep is controlled by both circadian and homeostatic mechanisms, and environmental factors such as light, temperature, and social stimuli are fundamental in shaping and confining sleep episodes into the correct time of the day. Among environmental cues, light seems to have a prominent function in modulating the timing of sleep during the 24 h and, in this review, we will discuss the role of light inputs in modulating the distribution of the fly sleep-wake cycles. This phenomenon is of growing interest in the modern society, where artificial light exposure during the night is a common trait, opening the possibility to study Drosophila as a model organism for investigating shift-work disorders.

CRY-dependent plasticity of tetrad presynaptic sites in the visual system of Drosophila at the morning peak of activity and sleep.

Tetrad synapses are formed between the retina photoreceptor terminals and postsynaptic cells in the first optic neuropil (lamina) of Drosophila. They are remodelled in the course of the day and show distinct functional changes during activity and sleep. These changes result from fast degradation of the presynaptic scaffolding protein Bruchpilot (BRP) by Cryptochrome (CRY) in the morning and depend on BRP-170, one of two BRP isoforms. This process also affects the number of synaptic vesicles, both clear and dense-core, delivered to the presynaptic elements. In cry01 mutants lacking CRY and in brpΔ170, the number of synaptic vesicles is lower in the morning peak of activity than during night-sleep while in wild-type flies the number of synaptic vesicles is similar at these two time points. CRY may also set phase of the circadian rhythm in plasticity of synapses. The process of synapse remodelling stimulates the formation of clear synaptic vesicles in the morning. They carry histamine, a neurotransmitter in tetrad synapses and seem to be formed from glial capitate projections inside the photoreceptor terminals. In turn dense-core vesicles probably carry synaptic proteins building the tetrad presynaptic element.

Corrigendum: One Actor, Multiple Roles: The Performances of Cryptochrome in Drosophila.

[This corrects the article DOI: 10.3389/fphys.2020.00099.].

Communication Among Photoreceptors and the Central Clock Affects Sleep Profile.

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 v s and l-LN v s 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 v s, which provides new light input to the clock network.

One Actor, Multiple Roles: The Performances of Cryptochrome in Drosophila.

Cryptochromes (CRYs) are flavoproteins that are sensitive to blue light, first identified in Arabidopsis and then in Drosophila and mice. They are evolutionarily conserved and play fundamental roles in the circadian clock of living organisms, enabling them to adapt to the daily 24-h cycles. The role of CRYs in circadian clocks differs among different species: in plants, they have a blue light-sensing activity whereas in mammals they act as light-independent transcriptional repressors within the circadian clock. These two different functions are accomplished by two principal types of CRYs, the light-sensitive plant/insect type 1 CRY and the mammalian type 2 CRY acting as a negative autoregulator in the molecular circadian clockwork. Drosophila melanogaster possesses just one CRY, belonging to type 1 CRYs. Nevertheless, this single CRY appears to have different functions, specific to different organs, tissues, and even subset of cells in which it is expressed. In this review, we will dissect the multiple roles of this single CRY in Drosophila, focusing on the regulatory mechanisms that make its pleiotropy possible.

New Drosophila Circadian Clock Mutants Affecting Temperature Compensation Induced by Targeted Mutagenesis of Timeless.

Drosophila melanogaster has served as an excellent genetic model to decipher the molecular basis of the circadian clock. Two key proteins, PERIOD (PER) and TIMELESS (TIM), are particularly well explored and a number of various arrhythmic, slow, and fast clock mutants have been identified in classical genetic screens. Interestingly, the free running period (tau, τ) is influenced by temperature in some of these mutants, whereas τ is temperature-independent in other mutant lines as in wild-type flies. This, so-called "temperature compensation" ability is compromised in the mutant timeless allele "ritsu" (tim rit ), and, as we show here, also in the tim blind allele, mapping to the same region of TIM. To test if this region of TIM is indeed important for temperature compensation, we generated a collection of new mutants and mapped functional protein domains involved in the regulation of τ and in general clock function. We developed a protocol for targeted mutagenesis of specific gene regions utilizing the CRISPR/Cas9 technology, followed by behavioral screening. In this pilot study, we identified 20 new timeless mutant alleles with various impairments of temperature compensation. Molecular characterization revealed that the mutations included short in-frame insertions, deletions, or substitutions of a few amino acids resulting from the non-homologous end joining repair process. Our protocol is a fast and cost-efficient systematic approach for functional analysis of protein-coding genes and promoter analysis in vivo. Interestingly, several mutations with a strong temperature compensation defect map to one specific region of TIM. Although the exact mechanism of how these mutations affect TIM function is as yet unknown, our in silico analysis suggests they affect a putative nuclear export signal (NES) and phosphorylation sites of TIM. Immunostaining for PER was performed on two TIM mutants that display longer τ at 25°C and complete arrhythmicity at 28°C. Consistently with the behavioral phenotype, PER immunoreactivity was reduced in circadian clock neurons of flies exposed to elevated temperatures.

Effects of MUL1 and PARKIN on the circadian clock, brain and behaviour in Drosophila Parkinson's disease models.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Transcriptome profiling reveals male- and female-specific gene expression pattern and novel gene candidates for the control of sex determination and gonad development in Xenopus laevis.

Xenopus laevis is an amphibian (frog) species widely used in developmental biology and genetics. To unravel the molecular machinery regulating sex differentiation of Xenopus gonads, we analyzed for the first time the transcriptome of developing amphibian gonads covering sex determination period. We applied microarray at four developmental stages: (i) NF50 (undifferentiated gonad during sex determination), (ii) NF53 (the onset of sexual differentiation of the gonads), (iii) NF56 (sexual differentiation of the gonads), and (iv) NF62 (developmental progression of differentiated gonads). Our analysis showed that during the NF50, the genetic female (ZW) gonads expressed more sex-specific genes than genetic male (ZZ) gonads, which suggests that a robust genetic program is realized during female sex determination in Xenopus. However, a contrasting expression pattern was observed at later stages (NF56 and NF62), when the ZW gonads expressed less sex-specific genes than ZZ gonads, i.e., more genes may be involved in further development of the male gonads (ZZ). We identified sexual dimorphism in the expression of several functional groups of genes, including signaling factors, proteases, protease inhibitors, transcription factors, extracellular matrix components, extracellular matrix enzymes, cell adhesion molecules, and epithelium-specific intermediate filaments. In addition, our analysis detected a sexually dimorphic expression of many uncharacterized genes of unknown function, which should be studied further to reveal their identity and if/how they regulate gonad development, sex determination, and sexual differentiation. Comparison between genes sex-specifically expressed in developing gonads of Xenopus and available transcriptome data from zebrafish, two reptile species, chicken, and mouse revealed significant differences in the genetic control of sex determination and gonad development. This shows that the genetic control of gonad development is evolutionarily malleable.

Overexpression of Mitochondrial Ligases Reverses Rotenone-Induced Effects in a Drosophila Model of Parkinson's Disease.

Mul1 and Park are two major mitochondrial ligases responsible for mitophagy. Damaged mitochondria that cannot be removed are a source of an increased level of free radicals, which in turn can destructively affect other cell organelles as well as entire cells. One of the toxins that damages mitochondria is rotenone, a neurotoxin that after exposure displays symptoms typical of Parkinson's disease. In the present study, we showed that overexpressing genes encoding mitochondrial ligases protects neurons during treatment with rotenone. Drosophila strains with overexpressed mul1 or park show a significantly reduced degeneration of dopaminergic neurons, as well as normal motor activity during exposure to rotenone. In the nervous system, rotenone affected synaptic proteins, including Synapsin, Synaptotagmin and Disk Large1, as well as the structure of synaptic vesicles, while high levels of Mul1 or Park suppressed degenerative events at synapses. We concluded that increased levels of mitochondrial ligases are neuroprotective and could be considered in developing new therapies for Parkinson's disease.