Bi-allelic genetic variants in the translational GTPases GTPBP1 and GTPBP2 cause a distinct identical neurodevelopmental syndrome.

The homologous genes GTPBP1 and GTPBP2 encode GTP-binding proteins 1 and 2, which are involved in ribosomal homeostasis. Pathogenic variants in GTPBP2 were recently shown to be an ultra-rare cause of neurodegenerative or neurodevelopmental disorders (NDDs). Until now, no human phenotype has been linked to GTPBP1. Here, we describe individuals carrying bi-allelic GTPBP1 variants that display an identical phenotype with GTPBP2 and characterize the overall spectrum of GTP-binding protein (1/2)-related disorders. In this study, 20 individuals from 16 families with distinct NDDs and syndromic facial features were investigated by whole-exome (WES) or whole-genome (WGS) sequencing. To assess the functional impact of the identified genetic variants, semi-quantitative PCR, western blot, and ribosome profiling assays were performed in fibroblasts from affected individuals. We also investigated the effect of reducing expression of CG2017, an ortholog of human GTPBP1/2, in the fruit fly Drosophila melanogaster. Individuals with bi-allelic GTPBP1 or GTPBP2 variants presented with microcephaly, profound neurodevelopmental impairment, pathognomonic craniofacial features, and ectodermal defects. Abnormal vision and/or hearing, progressive spasticity, choreoathetoid movements, refractory epilepsy, and brain atrophy were part of the core phenotype of this syndrome. Cell line studies identified a loss-of-function (LoF) impact of the disease-associated variants but no significant abnormalities on ribosome profiling. Reduced expression of CG2017 isoforms was associated with locomotor impairment in Drosophila. In conclusion, bi-allelic GTPBP1 and GTPBP2 LoF variants cause an identical, distinct neurodevelopmental syndrome. Mutant CG2017 knockout flies display motor impairment, highlighting the conserved role for GTP-binding proteins in CNS development across species.

Light triggers a network switch between circadian morning and evening oscillators controlling behaviour during daily temperature cycles.

Proper timing of rhythmic locomotor behavior is the consequence of integrating environmental conditions and internal time dictated by the circadian clock. Rhythmic environmental input like daily light and temperature changes (called Zeitgeber) reset the molecular clock and entrain it to the environmental time zone the organism lives in. Furthermore, depending on the absolute temperature or light intensity, flies exhibit their main locomotor activity at different times of day, i.e., environmental input not only entrains the circadian clock but also determines the phase of a certain behavior. To understand how the brain clock can distinguish between (or integrate) an entraining Zeitgeber and environmental effects on activity phase, we attempted to entrain the clock with a Zeitgeber different from the environmental input used for phasing the behavior. 150 clock neurons in the Drosophila melanogaster brain control different aspects of the daily activity rhythms and are organized in various clusters. During regular 12 h light: 12 h dark cycles at constant mild temperature (LD 25°C, LD being the Zeitgeber), so called morning oscillator (MO) neurons control the increase of locomotor activity just before lights-on, while evening oscillator (EO) neurons regulate the activity increase at the end of the day, a few hours before lights-off. Here, using 12 h: 12 h 25°C:16°C temperature cycles as Zeitgeber, we attempted to look at the impact of light on phasing locomotor behavior. While in constant light and 25°C:16°C temperature cycles (LLTC), flies show an unimodal locomotor activity peak in the evening, during the same temperature cycle, but in the absence of light (DDTC), the phase of the activity peak is shifted to the morning. Here, we show that the EO is necessary for synchronized behavior in LLTC but not for entraining the molecular clock of the other clock neuronal groups, while the MO controls synchronized morning activity in DDTC. Interestingly, our data suggest that the influence of the EO on the synchronization increases depending on the length of the photoperiod (constant light vs 12 h of light). Hence, our results show that effects of different environmental cues on clock entrainment and activity phase can be separated, allowing to decipher their integration by the circadian clock.

dyschronic, a Drosophila homolog of a deaf-blindness gene, regulates circadian output and Slowpoke channels.

Many aspects of behavior and physiology are under circadian control. In Drosophila, the molecular clock that regulates rhythmic patterns of behavior has been extensively characterized. In contrast, genetic loci involved in linking the clock to alterations in motor activity have remained elusive. In a forward-genetic screen, we uncovered a new component of the circadian output pathway, which we have termed dyschronic (dysc). dysc mutants exhibit arrhythmic locomotor behavior, yet their eclosion rhythms are normal and clock protein cycling remains intact. Intriguingly, dysc is the closest Drosophila homolog of whirlin, a gene linked to type II Usher syndrome, the leading cause of deaf-blindness in humans. Whirlin and other Usher proteins are expressed in the mammalian central nervous system, yet their function in the CNS has not been investigated. We show that DYSC is expressed in major neuronal tracts and regulates expression of the calcium-activated potassium channel SLOWPOKE (SLO), an ion channel also required in the circadian output pathway. SLO and DYSC are co-localized in the brain and control each other's expression post-transcriptionally. Co-immunoprecipitation experiments demonstrate they form a complex, suggesting they regulate each other through protein-protein interaction. Furthermore, electrophysiological recordings of neurons in the adult brain show that SLO-dependent currents are greatly reduced in dysc mutants. Our work identifies a Drosophila homolog of a deaf-blindness gene as a new component of the circadian output pathway and an important regulator of ion channel expression, and suggests novel roles for Usher proteins in the mammalian nervous system.

The E3 ubiquitin ligase CTRIP controls CLOCK levels and PERIOD oscillations in Drosophila.

In the Drosophila circadian clock, the CLOCK/CYCLE complex activates the period and timeless genes that negatively feedback on CLOCK/CYCLE activity. The 24-h pace of this cycle depends on the stability of the clock proteins. RING-domain E3 ubiquitin ligases have been shown to destabilize PERIOD or TIMELESS. Here we identify a clock function for the circadian trip (ctrip) gene, which encodes a HECT-domain E3 ubiquitin ligase. ctrip expression in the brain is mostly restricted to clock neurons and its downregulation leads to long-period activity rhythms in constant darkness. This altered behaviour is associated with high CLOCK levels and persistence of phosphorylated PERIOD during the subjective day. The control of CLOCK protein levels does not require PERIOD. Thus, CTRIP seems to regulate the pace of the oscillator by controlling the stability of both the activator and the repressor of the feedback loop.

A novel period mutation implicating nuclear export in temperature compensation of the Drosophila circadian clock.

Circadian clocks are self-sustained molecular oscillators controlling daily changes of behavioral activity and physiology. For functional reliability and precision, the frequency of these molecular oscillations must be stable at different environmental temperatures, known as "temperature compensation." Despite being an intrinsic property of all circadian clocks, this phenomenon is not well understood at the molecular level. Here, we use behavioral and molecular approaches to characterize a novel mutation in the period (per) clock gene of Drosophila melanogaster, which alters a predicted nuclear export signal (NES) of the PER protein and affects temperature compensation. We show that this new perI530A allele leads to progressively longer behavioral periods and clock oscillations with increasing temperature in both clock neurons and peripheral clock cells. While the mutant PERI530A protein shows normal circadian fluctuations and post-translational modifications at cool temperatures, increasing temperatures lead to both severe amplitude dampening and hypophosphorylation of PERI530A. We further show that PERI530A displays reduced repressor activity at warmer temperatures, presumably because it cannot inactivate the transcription factor CLOCK (CLK), indicated by temperature-dependent altered CLK post-translational modification in perI530A flies. With increasing temperatures, nuclear accumulation of PERI530A within clock neurons is increased, suggesting that wild-type PER is exported out of the nucleus at warm temperatures. Downregulating the nuclear export factor CRM1 also leads to temperature-dependent changes of behavioral rhythms, suggesting that the PER NES and the nuclear export of clock proteins play an important role in temperature compensation of the Drosophila circadian clock.

A natural timeless polymorphism allowing circadian clock synchronization in "white nights".

Daily temporal organisation offers a fitness advantage and is determined by an interplay between environmental rhythms and circadian clocks. While light:dark cycles robustly synchronise circadian clocks, it is not clear how animals experiencing only weak environmental cues deal with this problem. Like humans, Drosophila originate in sub-Saharan Africa and spread North up to the polar circle, experiencing long summer days or even constant light (LL). LL disrupts clock function, due to constant activation of CRYPTOCHROME, which induces degradation of the clock protein TIMELESS (TIM), but temperature cycles are able to overcome these deleterious effects of LL. We show here that for this to occur a recently evolved natural timeless allele (ls-tim) is required, encoding the less light-sensitive L-TIM in addition to S-TIM, the only form encoded by the ancient s-tim allele. We show that only ls-tim flies can synchronise their behaviour to semi-natural conditions typical for Northern European summers, suggesting that this functional gain is driving the Northward ls-tim spread.

Antagonistic Regulation of Circadian Output and Synaptic Development by JETLAG and the DYSCHRONIC-SLOWPOKE Complex.

Circadian output genes act downstream of the clock to promote rhythmic changes in behavior and physiology, yet their molecular and cellular functions are not well understood. Here we characterize an interaction between regulators of circadian entrainment, output, and synaptic development in Drosophila that influences clock-driven anticipatory increases in morning and evening activity. We previously showed the JETLAG (JET) E3 ubiquitin ligase resets the clock upon light exposure, whereas the PDZ protein DYSCHRONIC (DYSC) regulates circadian locomotor output and synaptic development. Surprisingly, we find that JET and DYSC antagonistically regulate synaptic development at the larval neuromuscular junction, and reduced JET activity rescues arrhythmicity of dysc mutants. Consistent with our prior finding that DYSC regulates SLOWPOKE (SLO) potassium channel expression, jet mutations also rescue circadian and synaptic phenotypes in slo mutants. Collectively, our data suggest that JET, DYSC, and SLO promote circadian output in part by regulating synaptic morphology.

DN1p or the "Fluffy" Cerberus of Clock Outputs.

Drosophila melanogaster is a powerful genetic model to study the circadian clock. Recently, three drosophilists received the Nobel Prize for their intensive past and current work on the molecular clockwork (Nobel Prize 2017). The Drosophila brain clock is composed of about 150 clock neurons distributed along the lateral and dorsal regions of the protocerebrum. These clock neurons control the timing of locomotor behaviors. In standard light-dark (LD) conditions (12-12 h and constant 25°C), flies present a bi-modal locomotor activity pattern controlled by the clock. Flies increase their movement just before the light-transitions, and these behaviors are therefore defined as anticipatory. Two neuronal oscillators control the morning and evening anticipation. Knowing that the molecular clock cycles in phase in all clock neurons in the brain in LD, how can we explain the presence of two behavioral activity peaks separated by 12 h? According to one model, the molecular clock cycles in phase in all clock neurons, but the neuronal activity cycles with a distinct phase in the morning and evening oscillators. An alternative model takes the environmental condition into consideration. One group of clock neurons, the dorso-posterior clock neurons DN1p, drive two peaks of locomotor activity in LD even though their neuronal activity cycles with the same phase (late night/early morning). Interestingly, the locomotor outputs they control differ in their sensitivity to light and temperature. Hence, they must drive outputs to different neuropil regions in the brain, which also receive different inputs. Since 2010 and the presentation of the first specific DN1p manipulations, many studies have been performed to understand the role of this group of neurons in controlling locomotor behaviors. Hence, we review what we know about this heterogeneous group of clock neurons and discuss the second model to explain how clock neurons that oscillate with the same phase can drive behaviors at different times of the day.

Temperature synchronization of the Drosophila circadian clock protein PERIOD is controlled by the TRPA channel PYREXIA.

Circadian clocks are endogenous molecular oscillators that temporally organize behavioral activity thereby contributing to the fitness of organisms. To synchronize the fly circadian clock with the daily fluctuations of light and temperature, these environmental cues are sensed both via brain clock neurons, and by light and temperature sensors located in the peripheral nervous system. Here we demonstrate that the TRPA channel PYREXIA (PYX) is required for temperature synchronization of the key circadian clock protein PERIOD. We observe a molecular synchronization defect explaining the previously reported defects of pyx mutants in behavioral temperature synchronization. Surprisingly, surgical ablation of pyx-mutant antennae partially rescues behavioral synchronization, indicating that antennal temperature signals are modulated by PYX function to synchronize clock neurons in the brain. Our results suggest that PYX protects antennal neurons from faulty signaling that would otherwise interfere with temperature synchronization of the circadian clock neurons in the brain.

Neurocalcin regulates nighttime sleep and arousal in Drosophila.

Sleep-like states in diverse organisms can be separated into distinct stages, each with a characteristic arousal threshold. However, the molecular pathways underlying different sleep stages remain unclear. The fruit fly, Drosophila melanogaster, exhibits consolidated sleep during both day and night, with night sleep associated with higher arousal thresholds compared to day sleep. Here we identify a role for the neuronal calcium sensor protein Neurocalcin (NCA) in promoting sleep during the night but not the day by suppressing nocturnal arousal and hyperactivity. We show that both circadian and light-sensing pathways define the temporal window in which NCA promotes sleep. Furthermore, we find that NCA promotes sleep by suppressing synaptic release from a dispersed wake-promoting neural network and demonstrate that the mushroom bodies, a sleep-regulatory center, are a module within this network. Our results advance the understanding of how sleep stages are genetically defined.

A Wake-Promoting Circadian Output Circuit in Drosophila.

Circadian clocks play conserved roles in gating sleep and wake states throughout the day-night cycle [1-5]. In the fruit fly Drosophila melanogaster, DN1p clock neurons have been reported to play both wake- and sleep-promoting roles [6-11], suggesting a complex coupling of DN1p neurons to downstream sleep and arousal centers. However, the circuit logic by which DN1p neurons modulate sleep remains poorly understood. Here, we show that DN1p neurons can be divided into two morphologically distinct subsets. Projections from one subset surround the pars intercerebralis, a previously defined circadian output region [12]. In contrast, the second subset also sends presynaptic termini to a visual processing center, the anterior optic tubercle (AOTU) [13]. Within the AOTU, we find that DN1p neurons inhibit a class of tubercular-bulbar (TuBu) neurons that act to promote consolidated sleep. These TuBu neurons in turn form synaptic connections with R neurons of the ellipsoid body, a region linked to visual feature detection, locomotion, spatial memory, and sleep homeostasis [14-17]. Our results define a second output arm from DN1p neurons and suggest a role for TuBu neurons as regulators of sleep drive.

Reconfiguration of a Multi-oscillator Network by Light in the Drosophila Circadian Clock.

The brain clock that drives circadian rhythms of locomotor activity relies on a multi-oscillator neuronal network. In addition to synchronizing the clock with day-night cycles, light also reformats the clock-driven daily activity pattern. How changes in lighting conditions modify the contribution of the different oscillators to remodel the daily activity pattern remains largely unknown. Our data in Drosophila indicate that light readjusts the interactions between oscillators through two different modes. We show that a morning s-LNv > DN1p circuit works in series, whereas two parallel evening circuits are contributed by LNds and other DN1ps. Based on the photic context, the master pacemaker in the s-LNv neurons swaps its enslaved partner-oscillator-LNd in the presence of light or DN1p in the absence of light-to always link up with the most influential phase-determining oscillator. When exposure to light further increases, the light-activated LNd pacemaker becomes independent by decoupling from the s-LNvs. The calibration of coupling by light is layered on a clock-independent network interaction wherein light upregulates the expression of the PDF neuropeptide in the s-LNvs, which inhibits the behavioral output of the DN1p evening oscillator. Thus, light modifies inter-oscillator coupling and clock-independent output-gating to achieve flexibility in the network. It is likely that the light-induced changes in the Drosophila brain circadian network could reveal general principles of adapting to varying environmental cues in any neuronal multi-oscillator system.

Regulation of sleep plasticity by a thermo-sensitive circuit in Drosophila.

Sleep is a highly conserved and essential behaviour in many species, including the fruit fly Drosophila melanogaster. In the wild, sensory signalling encoding environmental information must be integrated with sleep drive to ensure that sleep is not initiated during detrimental conditions. However, the molecular and circuit mechanisms by which sleep timing is modulated by the environment are unclear. Here we introduce a novel behavioural paradigm to study this issue. We show that in male fruit flies, onset of the daytime siesta is delayed by ambient temperatures above 29 °C. We term this effect Prolonged Morning Wakefulness (PMW). We show that signalling through the TrpA1 thermo-sensor is required for PMW, and that TrpA1 specifically impacts siesta onset, but not night sleep onset, in response to elevated temperatures. We identify two critical TrpA1-expressing circuits and show that both contact DN1p clock neurons, the output of which is also required for PMW. Finally, we identify the circadian blue-light photoreceptor CRYPTOCHROME as a molecular regulator of PMW, and propose a model in which the Drosophila nervous system integrates information encoding temperature, light, and time to dynamically control when sleep is initiated. Our results provide a platform to investigate how environmental inputs co-ordinately regulate sleep plasticity.

WIDE AWAKE mediates the circadian timing of sleep onset.

How the circadian clock regulates the timing of sleep is poorly understood. Here, we identify a Drosophila mutant, wide awake (wake), that exhibits a marked delay in sleep onset at dusk. Loss of WAKE in a set of arousal-promoting clock neurons, the large ventrolateral neurons (l-LNvs), impairs sleep onset. WAKE levels cycle, peaking near dusk, and the expression of WAKE in l-LNvs is Clock dependent. Strikingly, Clock and cycle mutants also exhibit a profound delay in sleep onset, which can be rescued by restoring WAKE expression in LNvs. WAKE interacts with the GABAA receptor Resistant to Dieldrin (RDL), upregulating its levels and promoting its localization to the plasma membrane. In wake mutant l-LNvs, GABA sensitivity is decreased and excitability is increased at dusk. We propose that WAKE acts as a clock output molecule specifically for sleep, inhibiting LNvs at dusk to promote the transition from wake to sleep.