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.

Synucleinopathies Take Their Toll: Are TLRs a Way to Go?

The misfolding and subsequent abnormal accumulation and aggregation of α-Synuclein (αSyn) as insoluble fibrils in Lewy bodies and Lewy neurites is the pathological hallmark of Parkinson's disease (PD) and several neurodegenerative disorders. A combination of environmental and genetic factors is linked to αSyn misfolding, among which neuroinflammation is recognized to play an important role. Indeed, a number of studies indicate that a Toll-like receptor (TLR)-mediated neuroinflammation might lead to a dopaminergic neural loss, suggesting that TLRs could participate in the pathogenesis of PD as promoters of immune/neuroinflammatory responses. Here we will summarize our current understanding on the mechanisms of αSyn aggregation and misfolding, focusing on the contribution of TLRs to the progression of α-synucleinopathies and speculating on their link with the non-motor disturbances associated with aging and neurodegenerative disorders.

Mitochondrial autophagy in the sleeping brain.

A significant percentage of the mitochondrial mass is replaced on a daily basis via mechanisms of mitochondrial quality control. Through mitophagy (a selective type of autophagy that promotes mitochondrial proteostasis) cells keep a healthy pool of mitochondria, and prevent oxidative stress and inflammation. Furthermore, mitophagy helps adapting to the metabolic demand of the cells, which changes on a daily basis. Core components of the mitophagy process are PINK1 and Parkin, which mutations are linked to Parkinson's Disease. The crucial role of PINK1/Parkin pathway during stress-induced mitophagy has been extensively studied in vitro in different cell types. However, recent advances in the field allowed discovering that mitophagy seems to be only slightly affected in PINK1 KO mice and flies, putting into question the physiological relevance of this pathway in vivo in the whole organism. Indeed, several cell-specific PINK1/Parkin-independent mitophagy pathways have been recently discovered, which appear to be activated under physiological conditions such as those that promote mitochondrial proteome remodeling during differentiation or in response to specific physiological stimuli. In this Mini Review we want to summarize the recent advances in the field, and add another level of complexity by focusing attention on a potentially important aspect of mitophagy regulation: the implication of the circadian clock. Recent works showed that the circadian clock controls many aspects of mitochondrial physiology, including mitochondrial morphology and dynamic, respiratory activity, and ATP synthesis. Furthermore, one of the essential functions of sleep, which is controlled by the clock, is the clearance of toxic metabolic compounds from the brain, including ROS, via mechanisms of proteostasis. Very little is known about a potential role of the clock in the quality control mechanisms that maintain the mitochondrial repertoire healthy during sleep/wake cycles. More importantly, it remains completely unexplored whether (dys)function of mitochondrial proteostasis feedbacks to the circadian clockwork.

Arginine kinase interacts with 2MIT and is involved in Drosophila melanogaster short-term memory.

Mushroom bodies are a higher order center for sensory integration, learning and memory of the insect brain. Memory is generally subdivided into different phases. In the model organism Drosophila melanogaster, mushroom bodies have been shown to play a central role in both short- and long-term memory. In D. melanogaster, the gene 2mit codes a transmembrane protein carrying an extracellular Leucin-rich-repeat domain, which is highly transcribed in the mushroom and ellipsoid bodies of the adult fly brain and has a role in the early phase of memory. Utilizing coimmunoprecipitation experiments and mass spectrometry analyses, we have shown that 2MIT interacts with Arginine kinase in adult fly heads. Arginine kinase belongs to the family of Phosphagen kinases and plays a fundamental role in energy homeostasis. Using the GAL4/UAS binary system, we demonstrated that a downregulation of Arginine kinase mainly driven in the mushroom bodies affects short-term memory of Drosophila adult flies, in a courtship conditioning paradigm. As 2mit c03963 hypomorphic mutants showed comparable results when analyzed with the same assay, these data suggest that 2MIT and Arginine kinase are both involved in the same memory phenotype, likely interacting at the level of mushroom bodies. 2MIT and Arginine kinase are conserved among insects, the implications of which, along with their potential roles in other insect taxa are also discussed.

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.

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

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

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.

Peptidergic signaling from clock neurons regulates reproductive dormancy in Drosophila melanogaster.

With the approach of winter, many insects switch to an alternative protective developmental program called diapause. Drosophila melanogaster females overwinter as adults by inducing a reproductive arrest that is characterized by inhibition of ovarian development at previtellogenic stages. The insulin producing cells (IPCs) are key regulators of this process, since they produce and release insulin-like peptides that act as diapause-antagonizing hormones. Here we show that in D. melanogaster two neuropeptides, Pigment Dispersing Factor (PDF) and short Neuropeptide F (sNPF) inhibit reproductive arrest, likely through modulation of the IPCs. In particular, genetic manipulations of the PDF-expressing neurons, which include the sNPF-producing small ventral Lateral Neurons (s-LNvs), modulated the levels of reproductive dormancy, suggesting the involvement of both neuropeptides. We expressed a genetically encoded cAMP sensor in the IPCs and challenged brain explants with synthetic PDF and sNPF. Bath applications of both neuropeptides increased cAMP levels in the IPCs, even more so when they were applied together, suggesting a synergistic effect. Bath application of sNPF additionally increased Ca2+ levels in the IPCs. Our results indicate that PDF and sNPF inhibit reproductive dormancy by maintaining the IPCs in an active state.

The RNA Helicase BELLE Is Involved in Circadian Rhythmicity and in Transposons Regulation in Drosophila melanogaster.

Circadian clocks control and synchronize biological rhythms of several behavioral and physiological phenomena in most, if not all, organisms. Rhythm generation relies on molecular auto-regulatory oscillations of interlocked transcriptional-translational feedback loops. Rhythmic clock-gene expression is at the base of rhythmic protein accumulation, though post-transcriptional and post-translational mechanisms have evolved to adjust and consolidate the proper pace of the clock. In Drosophila, BELLE, a conserved DEAD-box RNA helicase playing important roles in reproductive capacity, is involved in the small RNA-mediated regulation associated to the piRNA pathway. Here, we report that BELLE is implicated in the circadian rhythmicity and in the regulation of endogenous transposable elements (TEs) in both nervous system and gonads. We suggest that BELLE acts as important element in the piRNA-mediated regulation of the TEs and raise the hypothesis that this specific regulation could represent another level of post-transcriptional control adopted by the clock to ensure the proper rhythmicity.

Morning Bright Light Treatment for Sleep-Wake Disturbances in Primary Biliary Cholangitis: A Pilot Study.

Patients with Primary Biliary Cholangitis (PBC) exhibit delayed sleep-wake habits, disturbed night sleep and daytime sleepiness/fatigue. Such combination of symptoms is reminiscent of delayed sleep-wake phase disorder (DSPD), which benefits from morning light treatment. The aim of the present pilot study was to test the effect of morning light treatment in a group of 13 well-characterized patients with PBC [all females; (mean ± SD) 53 ± 10 years]. Six healthy individuals (4 females, 57 ± 14 years) and 7 patients with cirrhosis (1 female, 57 ± 12 years) served as controls and diseased controls, respectively. At baseline, all participants underwent an assessment of quality of life, diurnal preference, sleep quality/timing (subjective plus actigraphy), daytime sleepiness, and urinary 6-sulphatoxymelatonin (aMT6s) rhythmicity. Then they underwent a 15-day course of morning bright light treatment, immediately after getting up (light box, 10,000 lux, 45 min) whilst monitoring sleep-wake patterns and aMT6s rhythmicity. At baseline, both patients with PBC and patients with cirrhosis had significantly worse subjective sleep quality compared to controls. In patients with PBC, light treatment resulted in an improvement in subjective sleep quality and a reduction in daytime sleepiness. In addition, both their sleep onset and get-up time were significantly advanced. Finally, the robustness of aMT6s rhythmicity (i.e., strength of the cosinor fit) increased after light administration but post-hoc comparisons were not significant in any of the groups. In conclusion, a brief course of morning bright light treatment had positive effects on subjective sleep quality, daytime sleepiness, and sleep timing in patients with PBC. This unobtrusive, side-effect free, non-pharmacological treatment is worthy of further study.

Calmodulin Enhances Cryptochrome Binding to INAD in Drosophila Photoreceptors.

Light is the main environmental stimulus that synchronizes the endogenous timekeeping systems in most terrestrial organisms. Drosophila cryptochrome (dCRY) is a light-responsive flavoprotein that detects changes in light intensity and wavelength around dawn and dusk. We have previously shown that dCRY acts through Inactivation No Afterpotential D (INAD) in a light-dependent manner on the Signalplex, a multiprotein complex that includes visual-signaling molecules, suggesting a role for dCRY in fly vision. Here, we predict and demonstrate a novel Ca2+-dependent interaction between dCRY and calmodulin (CaM). Through yeast two hybrid, coimmunoprecipitation (Co-IP), nuclear magnetic resonance (NMR) and calorimetric analyses we were able to identify and characterize a CaM binding motif in the dCRY C-terminus. Similarly, we also detailed the CaM binding site of the scaffold protein INAD and demonstrated that CaM bridges dCRY and INAD to form a ternary complex in vivo. Our results suggest a process whereby a rapid dCRY light response stimulates an interaction with INAD, which can be further consolidated by a novel mechanism regulated by CaM.

Cryptochrome Interacts With Actin and Enhances Eye-Mediated Light Sensitivity of the Circadian Clock in Drosophila melanogaster.

Cryptochromes (CRYs) are a class of flavoproteins that sense blue light. In animals, CRYs are expressed in the eyes and in the clock neurons that control sleep/wake cycles and are implied in the generation and/or entrainment of circadian rhythmicity. Moreover, CRYs are sensing magnetic fields in insects as well as in humans. Here, we show that in the fruit fly Drosophila melanogaster CRY plays a light-independent role as "assembling" protein in the rhabdomeres of the compound eyes. CRY interacts with actin and appears to increase light sensitivity of the eyes by keeping the "signalplex" of the phototransduction cascade close to the membrane. By this way, CRY also enhances light-responses of the circadian clock.

Modulation of miR-210 alters phasing of circadian locomotor activity and impairs projections of PDF clock neurons in Drosophila melanogaster.

Single microRNAs are usually associated with hundreds of putative target genes that can influence multiple phenotypic traits in Drosophila, ranging from development to behaviour. We investigated the function of Drosophila miR-210 in circadian behaviour by misexpressing it within circadian clock cells. Manipulation of miR-210 expression levels in the PDF (pigment dispersing factor) positive neurons affected the phase of locomotor activity, under both light-dark conditions and constant darkness. PER cyclical expression was not affected in clock neurons, however, when miR-210 was up-regulated, a dramatic alteration in the morphology of PDF ventral lateral neuron (LNv) arborisations was observed. The effect of miR-210 in shaping neuronal projections was confirmed in vitro, using a Drosophila neuronal cell line. A transcriptomic analysis revealed that miR-210 overexpression affects the expression of several genes belonging to pathways related to circadian processes, neuronal development, GTPases signal transduction and photoreception. Collectively, these data reveal the role of miR-210 in modulating circadian outputs in flies and guiding/remodelling PDF positive LNv arborisations and indicate that miR-210 may have pleiotropic effects on the clock, light perception and neuronal development.

A Semi-natural Approach for Studying Seasonal Diapause in Drosophila melanogaster Reveals Robust Photoperiodicity.

The fruit fly Drosophila melanogaster survives thermally stressful conditions in a state of reproductive dormancy (diapause), manifested by reduced metabolic activity and arrested ovarian development in females. Unlike insects that rely primarily on photoperiodic stimuli to initiate the diapause program, in this species dormancy is regulated by low temperature and enhanced by shorter photoperiods. Overwintering phenotypes are usually studied under simple laboratory conditions, where animals are exposed to rectangular light-dark (LD) cycles at a constant temperature. We sought to adopt more realistic diapause protocols by generating LD profiles that better mimic outdoor conditions. Experimental flies were subjected to semi-natural late autumn and summer days, while control females received the same amounts of light but in rectangular LD cycles (LD 8:16 and LD 15:9, respectively). We observed that semi-natural autumnal days induced a higher proportion of females to enter dormancy, while females in semi-natural summer days showed reduced diapause compared with their corresponding rectangular controls, generating an impressive photoperiodic response. In contrast, under rectangular light regimes, the diapause of Drosophila field lines exhibited minimal photoperiodicity. Our semi-natural method reveals that D. melanogaster diapause is considerably more photoperiodic than previously believed and suggests that this seasonal response is best studied under simulated natural lighting conditions.

Functional characterization of the circadian clock in the Antarctic krill, Euphausia superba.

Antarctic krill (Euphausia superba) is a key species in Southern Ocean ecosystem where it plays a central role in the Antarctic food web. Available information supports the existence of an endogenous timing system in krill enabling it to synchronize metabolism and behavior with an environment characterized by extreme seasonal changes in terms of day length, food availability, and surface ice extent. A screening of our transcriptome database "KrillDB" allowed us to identify the putative orthologues of 20 circadian clock components. Mapping of conserved domains and phylogenetic analyses strongly supported annotations of the identified sequences. Luciferase assays and co-immunoprecipitation experiments allowed us to define the role of the main clock components. Our findings provide an overall picture of the molecular mechanisms underlying the functioning of the endogenous circadian clock in the Antarctic krill and shed light on their evolution throughout crustaceans speciation. Interestingly, the core clock machinery shows both mammalian and insect features that presumably contribute to an evolutionary strategy to cope with polar environment's challenges. Moreover, despite the extreme variability characterizing the Antarctic seasonal day length, the conserved light mediated degradation of the photoreceptor EsCRY1 suggests a persisting pivotal role of light as a Zeitgeber.

Cryptochrome Is a Regulator of Synaptic Plasticity in the Visual System of Drosophila melanogaster.

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 2 hybrid (Y2H) and in situ proximity ligation assay (PLA) 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 opsin repertoire of the Antarctic krill Euphausia superba.

The Antarctic krill Euphausia superba experiences almost all marine photic environments throughout its life cycle. Antarctic krill eggs hatch in the aphotic zone up to 1000m depth and larvae develop on their way to the ocean surface (development ascent) and are exposed to different quality (wavelength) and quantity (irradiance) of light. Adults show a daily vertical migration pattern, moving downward during the day and upward during the night within the top 200m of the water column. Seawater acts as a potent chromatic filter and animals have evolved different opsin photopigments to perceive photons of specific wavelengths. We have investigated the transcriptome of E. superba and, using a candidate gene approach, we identified six novel opsins. Five are r-type visual opsins: four middle-wavelength-sensitive (EsRh2, EsRh3, EsRh4 and EsRh5) and one long-wavelength-sensitive (EsRh6). Moreover, we have identified a non-visual opsin, the EsPeropsin. All these newly identified opsin genes were significantly expressed in compound eyes and brain, while only EsPeropsin and EsRh2 were clearly detected also in the abdomen. A temporal modulation in the transcription of these novel opsins was found, but statistically significant oscillations were only observed in EsRrh3 and EsPeropsin. Our results contribute to the dissection of the complex photoreception system of E. superba, which enables this species to respond to the daily and seasonal changes in irradiance and spectral composition in the Southern Ocean.

Isoform-specific interactions of the von Hippel-Lindau tumor suppressor protein.

Deregulation of the von Hippel-Lindau tumor suppressor protein (pVHL) is considered one of the main causes for malignant renal clear-cell carcinoma (ccRCC) insurgence. In human, pVHL exists in two isoforms, pVHL19 and pVHL30 respectively, displaying comparable tumor suppressor abilities. Mutations of the p53 tumor suppressor gene have been also correlated with ccRCC insurgence and ineffectiveness of treatment. A recent proteomic analysis linked full length pVHL30 with p53 pathway regulation through complex formation with the p14ARF oncosuppressor. The alternatively spliced pVHL19, missing the first 53 residues, lacks this interaction and suggests an asymmetric function of the two pVHL isoforms. Here, we present an integrative bioinformatics and experimental characterization of the pVHL oncosuppressor isoforms. Predictions of the pVHL30 N-terminus three-dimensional structure suggest that it may exist as an ensemble of structured and disordered forms. The results were used to guide Yeast two hybrid experiments to highlight isoform-specific binding properties. We observed that the physical pVHL/p14ARF interaction is specifically mediated by the 53 residue long pVHL30 N-terminal region, suggesting that this N-terminus acts as a further pVHL interaction interface. Of note, we also observed that the shorter pVHL19 isoform shows an unexpected high tendency to form homodimers, suggesting an additional isoform-specific binding specialization.

period and timeless mRNA Splicing Profiles under Natural Conditions in Drosophila melanogaster.

Previous analysis of Drosophila circadian behavior under natural conditions has revealed a number of novel and unexpected features. Here we focus on the oscillations of per and tim mRNAs and their posttranscriptional regulation and observe significant differences in molecular cycling under laboratory and natural conditions. In particular, robust per mRNA cycling from fly heads is limited to the summers, whereas tim RNA cycling is observed throughout the year. When both transcripts do cycle, their phases are similar, except for the very warmest summer months. We also study the natural splicing profiles of per and tim transcripts and observe a clear relationship between temperature and splicing. In natural conditions, we confirm the relationship between accumulation of the per(spliced) variant, low temperature, and the onset of the evening component of locomotor activity, first described in laboratory conditions. Intriguingly, in the case of tim splicing, we detect the opposite relationship, with tim(spliced) expression increasing at higher temperatures. A first characterization of the 4 different TIM protein isoforms (resulting from the combination of the natural N-terminus length polymorphism and the C-terminus alternative splicing) using the 2-hybrid assay showed that the TIM(unspliced) isoforms have a stronger affinity for CRY, but not for PER, suggesting that the tim 3' splicing could have physiological significance, possibly in temperature entrainment and/or adaptation to seasonal environments.