Restoration of Sleep and Circadian Behavior by Autophagy Modulation in Huntington's Disease.

Circadian and sleep defects are well documented in Huntington's disease (HD). Modulation of the autophagy pathway has been shown to mitigate toxic effects of mutant Huntingtin (HTT) protein. However, it is not clear whether autophagy induction can also rescue circadian and sleep defects. Using a genetic approach, we expressed human mutant HTT protein in a subset of Drosophila circadian neurons and sleep center neurons. In this context, we examined the contribution of autophagy in mitigating toxicity caused by mutant HTT protein. We found that targeted overexpression of an autophagy gene, Atg8a in male flies, induces autophagy pathway and partially rescues several HTT-induced behavioral defects, including sleep fragmentation, a key hallmark of many neurodegenerative disorders. Using cellular markers and genetic approaches, we demonstrate that indeed the autophagy pathway is involved in behavioral rescue. Surprisingly, despite behavioral rescue and evidence for the involvement of the autophagy pathway, the large visible aggregates of mutant HTT protein were not eliminated. We show that the rescue in behavior is associated with increased mutant protein aggregation and possibly enhanced output from the targeted neurons, resulting in the strengthening of downstream circuits. Overall, our study suggests that, in the presence of mutant HTT protein, Atg8a induces autophagy and improves the functioning of circadian and sleep circuits.SIGNIFICANCE STATEMENT Defects in sleep and circadian rhythms are well documented in Huntington's disease. Recent literature suggests that circadian and sleep disturbances can exacerbate neurodegenerative phenotypes. Hence, identifying potential modifiers that can improve the functioning of these circuits could greatly improve disease management. We used a genetic approach to enhance cellular proteostasis and found that overexpression of a crucial autophagy gene, Atg8a, induces the autophagy pathway in the Drosophila circadian and sleep neurons and rescues sleep and activity rhythm. We demonstrate that the Atg8a improves synaptic function of these circuits by possibly enhancing the aggregation of the mutant protein in neurons. Further, our results suggest that differences in basal levels of protein homeostatic pathways is a factor that determines selective susceptibility of neurons.

Reorganization of circadian activity and the pacemaker circuit under novel light regimes.

Many environmental features are cyclic, with predictable changes across the day, seasons and latitudes. Additionally, anthropogenic, artificial-light-induced changes in photoperiod or shiftwork-driven novel light/dark cycles also occur. Endogenous timekeepers or circadian clocks help organisms cope with such changes. The remarkable plasticity of clocks is evident in the waveforms of behavioural and molecular rhythms they govern. Despite detailed mechanistic insights into the functioning of the circadian clock, practical means to manipulate activity waveform are lacking. Previous studies using a nocturnal rodent model showed that novel light regimes caused locomotor activity to bifurcate such that mice showed two bouts of activity restricted to the dimly lit phases. Here, we explore the generalizability of these findings and leverage the genetic toolkit of Drosophila melanogaster to obtain mechanistic insights into this unique phenomenon. We find that dim scotopic illumination of specific durations induces circadian photoreceptor CRYPTOCHROME-dependent activity bifurcation in male flies. We show circadian reorganization of the pacemaker circuit, wherein the 'evening' neurons regulate the timing of both bouts of activity under novel light regimes. Our findings indicate that such environmental regimes can be exploited to design light cycles, which can ease the circadian waveform into synchronizing with challenging conditions.

Mechanosensory Stimulation via Nanchung Expressing Neurons Can Induce Daytime Sleep in Drosophila.

The neuronal and genetic bases of sleep, a phenomenon considered crucial for well-being of organisms, has been under investigation using the model organism Drosophila melanogaster Although sleep is a state where sensory threshold for arousal is greater, it is known that certain kinds of repetitive sensory stimuli, such as rocking, can indeed promote sleep in humans. Here we report that orbital motion-aided mechanosensory stimulation promotes sleep of male and female Drosophila, independent of the circadian clock, but controlled by the homeostatic system. Mechanosensory receptor nanchung (Nan)-expressing neurons in the chordotonal organs mediate this sleep induction: flies in which these neurons are either silenced or ablated display significantly reduced sleep induction on mechanosensory stimulation. Transient activation of the Nan-expressing neurons also enhances sleep levels, confirming the role of these neurons in sleep induction. We also reveal that certain regions of the antennal mechanosensory and motor center in the brain are involved in conveying information from the mechanosensory structures to the sleep centers. Thus, we show, for the first time, that a circadian clock-independent pathway originating from peripherally distributed mechanosensors can promote daytime sleep of flies Drosophila melanogasterSIGNIFICANCE STATEMENT Our tendency to fall asleep in moving vehicles or the practice of rocking infants to sleep suggests that slow rhythmic movement can induce sleep, although we do not understand the mechanistic basis of this phenomenon. We find that gentle orbital motion can induce behavioral quiescence even in flies, a highly genetically tractable system for sleep studies. We demonstrate that this is indeed true sleep based on its rapid reversibility by sensory stimulation, enhanced arousal threshold, and homeostatic control. Furthermore, we demonstrate that mechanosensory neurons expressing a TRPV channel nanchung, located in the antennae and chordotonal organs, mediate orbital motion-induced sleep by communicating with antennal mechanosensory motor centers, which in turn may project to sleep centers in the brain.

Responses of activity rhythms to temperature cues evolve in Drosophila populations selected for divergent timing of eclosion.

Even though the rhythms in adult emergence and locomotor activity are two different phenomena that occur at distinct life stages of the fly life cycle, previous studies have hinted at similarities in certain aspects of the organisation of the circadian clock driving these two rhythms. For instance, the period gene plays an important regulatory role in both rhythms. In an earlier study, we have shown that selection on timing of adult emergence behaviour in populations of Drosophila melanogaster leads to the co-evolution of temperature sensitivity of circadian clocks driving eclosion. In this study, we investigated whether temperature sensitivity of the locomotor activity rhythm evolved in our populations separately from the adult emergence rhythm, with the goal of understanding the extent of similarity (or lack thereof) in circadian organisation underlying the two rhythms. We found that in response to simulated jetlag with temperature cycles, late chronotypes (populations selected for predominant emergence during dusk) indeed re-entrained faster than early chronotypes (populations selected for predominant emergence during dawn) to 6 h phase delays, thereby indicating enhanced sensitivity of the activity/rest clock to temperature cues in these stocks (entrainment is the synchronisation of internal rhythms to cyclic environmental time cues). Additionally, we found that late chronotypes show higher plasticity of phases across regimes, day-to-day stability in phases and amplitude of entrainment, all indicative of enhanced temperature-sensitive activity/rest rhythms. Our results highlight remarkably similar organisation principles between circadian clocks regulating emergence and activity/rest rhythms.

Environmental cycles regulate development time via circadian clock mediated gating of adult emergence.

BACKGROUND: Previous studies have implicated a role for circadian clocks in regulating pre-adult development of organisms. Among them two approaches are most notable: 1) use of insects whose clocks have different free-running periods and 2) imposition of artificial selection on either rate of development, timing of emergence or circadian period in laboratory populations. Using these two approaches, influence of clock on rate of development has been elucidated. However, the contribution of circadian clocks in determining time taken for pre-adult development has remained unclear. Here we present results of our studies aimed to understand this influence by examining populations of fruit flies carrying three different alleles of the period gene and hence having different free-running periods. We tried to achieve similarity of genetic background among the three strains while also ensuring that they harbored sufficient variation on loci other than period gene. RESULTS: We find that under constant conditions, flies with long period have slower development whereas in presence of light-dark cycles (LD) of various lengths, the speed of development for each genotype is influenced by whether their eclosion rhythms can entrain to them. Under LD 12:12 (T24), where all three strains entrain, they do not show any difference in time taken for emergence, whereas under LD 10:10 (T20) where long period flies do not entrain and LD 14:14 (T28) where short period flies do not entrain, they have slower and faster pre-adult development, respectively, compared to the controls. We also show that a prior stage in development namely pupation is not rhythmic though time taken for pupation is determined by both the environmental cycle and period allele. CONCLUSION: We discuss how in presence of daily time cues, interaction of the cyclic environmental factors with the clock determines the position and width of the gate available for a fly to emerge (duration of time within a cycle when adult emergence can occur) resulting in an altered developmental duration from that observed under constant conditions. We also discuss the relevance of genetic background influencing this regulation.

Synthetic Host-Guest Assembly in Cells and Tissues: Fast, Stable, and Selective Bioorthogonal Imaging via Molecular Recognition.

Bioorthogonal strategies are continuing to pave the way for new analytical tools in biology. Although a significant amount of progress has been made in developing covalent reaction based bioorthogonal strategies, balanced reactivity, and stability are often difficult to achieve from these systems. Alternatively, despite being kinetically beneficial, the development of noncovalent approaches that utilize fully synthetic and stable components remains challenging due to the lack of selectivity in conventional noncovalent interactions in the living cellular environment. Herein, we introduce a bioorthogonal assembly strategy based on a synthetic host-guest system featuring Cucurbit[7]uril (CB[7]) and adamantylamine (ADA). We demonstrate that highly selective and ultrastable host-guest interaction between CB[7] and ADA provides a noncovalent mechanism for assembling labeling agents, such as fluorophores and DNA, in cells and tissues for bioorthogonal imaging of molecular targets. Additionally, by combining with covalent reaction, we show that this CB[7]-ADA based noncovalent interaction enables simultaneous bioorthogonal labeling and multiplexed imaging in cells as well as tissue sections. Finally, we show that interaction between CB[7] and ADA fulfills the demands of specificity and stability that is required for assembling molecules in the complexities of a living cell. We demonstrate this by sensitive detection of metastatic cancer-associated cell surface protein marker as well as by showing the distribution and dynamics of F-actin in living cells.

Sleep deprivation negatively impacts reproductive output in Drosophila melanogaster.

Most animals sleep or exhibit a sleep-like state, yet the adaptive significance of this phenomenon remains unclear. Although reproductive deficits are associated with lifestyle-induced sleep deficiencies, how sleep loss affects reproductive physiology is poorly understood, even in model organisms. We aimed to bridge this mechanistic gap by impairing sleep in female fruit flies and testing its effect on egg output. We found that sleep deprivation by feeding caffeine or by mechanical perturbation resulted in decreased egg output. Transient activation of wake-promoting dopaminergic neurons decreased egg output in addition to sleep levels, thus demonstrating a direct negative impact of sleep deficit on reproductive output. Similarly, loss-of-function mutation in dopamine transporter fumin (fmn) led to both significant sleep loss and lowered fecundity. This demonstration of a direct relationship between sleep and reproductive fitness indicates a strong driving force for the evolution of sleep.

Significance of activity peaks in fruit flies, Drosophila melanogaster, under seminatural conditions.

Studies on circadian entrainment have traditionally been performed under controlled laboratory conditions. Although these studies have served the purpose of providing a broad framework for our understanding of regulation of rhythmic behaviors under cyclic conditions, they do not reveal how organisms keep time in nature. Although a few recent studies have attempted to address this, it is not yet clear which environmental factors regulate rhythmic behaviors in nature and how. Here, we report the results of our studies aimed at examining (i) whether and how changes in natural light affect activity/rest rhythm and (ii) what the functional significance of this rhythmic behavior might be. We found that wild-type strains of fruit flies, Drosophila melanogaster, display morning (M), afternoon (A), and evening (E) peaks of activity under seminatural conditions (SN), whereas under constant darkness in otherwise SN, they exhibited M and E peaks, and under constant light in SN, only the E peak occurred. Unlike the A peak, which requires exposure to bright light in the afternoon, light information is dispensable for the M and E peaks. Visual examination of behaviors suggests that the M peak is associated with courtship-related locomotor activity and the A peak is due to an artifact of the experimental protocol and largely circadian clock independent.

Simulating natural light and temperature cycles in the laboratory reveals differential effects on activity/rest rhythm of four Drosophilids.

Recent studies under semi-natural conditions have revealed various unique features of activity/rest rhythms in Drosophilids that differ from those under standard laboratory conditions. An additional afternoon peak (A-peak) has been reported for Drosophila melanogaster and another species D. malerkotliana while D. ananassae exhibited mostly unimodal diurnal activity. To tease apart the role of light and temperature in mediating these species-specific behaviours of four Drosophilid species D. melanogaster, D. malerkotliana, D. ananassae, and Zaprionus indianus we simulated gradual natural light and/or temperature cycles conditions in laboratory. The pattern observed under semi-natural conditions could be reproduced in the laboratory for all the species under a variety of simulated conditions. D. melanogaster and D. malerkotliana showed similar patterns where as D. ananassae consistently exhibited predominant morning activity under almost all regimes. Z. indianus showed clearly rhythmic activity mostly when temperature cycles were provided. We find that gradually changing light intensities reaching a sufficiently high peak value can elicit A-peak in D. melanogaster, D. malerkotliana, and D. ananassae even at mild ambient temperature. Furthermore, we show that high mid-day temperature could induce A-peak in all species even under constant light conditions suggesting that this A-peak is likely to be a stress response.

Drosophila melanogaster Neuromuscular Junction as a Model to Study Synaptopathies and Neuronal Autophagy.

Neuronal synapse dysfunction is a key characteristic of several neurodegenerative disorders, such as Alzheimer's disease, spinocerebellar ataxias, and Huntington's disease. Modeling these disorders to study synaptic dysfunction requires a robust and reproducible method for assaying the subtle changes associated with synaptopathies in terms of structure and function of the synapses. Drosophila melanogaster neuromuscular junctions (NMJs) serve as good models to study such alterations. Further, modifications in the microenvironment of synapses can sometimes reflect in the behavior of the animal, which can also be assayed in a high-throughput manner. The methods outlined in this chapter highlight assays to study the behavioral changes associated with synaptic dysfunction in a spinocerebellar ataxia type 3 (SCA3) model. Further, molecular assessment of alterations in NMJ structure and function is also summarized, followed by effects of autophagy pathway upregulation in providing neuroprotection. These methods can be further extended and modified to study the therapeutic effects of drugs or small molecules in providing neuroprotection for any synaptopathy models.

Lessons from sleeping flies: insights from Drosophila melanogaster on the neuronal circuitry and importance of sleep.

Sleep is a highly conserved behavior whose role is as yet unknown, although it is widely acknowledged as being important. Here we provide an overview of many vital questions regarding this behavior, that have been addressed in recent years using the genetically tractable model organism Drosophila melanogaster in several laboratories around the world. Rest in D. melanogaster has been compared to mammalian sleep and its homeostatic and circadian regulation have been shown to be controlled by intricate neuronal circuitry involving circadian clock neurons, mushroom bodies, and pars intercerebralis, although their exact roles are not entirely clear. We draw attention to the yet unanswered questions and contradictions regarding the nature of the interactions between the brain regions implicated in the control of sleep. Dopamine, octopamine, γ-aminobutyric acid (GABA), and serotonin are the chief neurotransmitters identified as functioning in different limbs of this circuit, either promoting arousal or sleep by modulating membrane excitability of underlying neurons. Some studies have suggested that certain brain areas may contribute towards both sleep and arousal depending on activation of specific subsets of neurons. Signaling pathways implicated in the sleep circuit include cyclic adenosine monophosphate (cAMP) and epidermal growth factor receptor-extracellular signal-regulated kinase (EGFR-ERK) signaling pathways that operate on different neural substrates. Thus, this field of research appears to be on the cusp of many new and exciting findings that may eventually help in understanding how this complex physiological phenomenon is modulated by various neuronal circuits in the brain. Finally, some efforts to approach the "Holy Grail" of why we sleep have been summarized.

Insights into differential activity patterns of drosophilids under semi-natural conditions.

We showed recently that Drosophila ananassae, a closely related and sympatric species of the commonly studied fruitfly D. melanogaster, shows distinctly deviant patterns in circadian activity/rest rhythm from the latter under a variety of laboratory conditions. To examine whether such differences extend to more natural conditions where a variety of time cues and similar environmental pressures might force different species to adopt similar temporal patterns, we examined these two species under semi-natural conditions over a span of 1.5 years. Furthermore, we asked to what extent features of activity/rest rhythm of flies are conserved across species under changing environmental conditions encountered across seasons, and to do so, we studied two more drosophilid species. We found that while each species exhibits seasonality in activity patterns, this seasonality is marked by interesting inter-specific differences. Similar to laboratory studies, D. ananassae showed activity mostly during the day, while D. melanogaster and D. malerkotliana exhibited almost similar activity patterns across seasons, with predominantly two peaks of activity, one in the morning and another in the evening. Throughout the year, Zaprionus indianus displayed very low levels of activity compared with D. melanogaster, yet, compared with those seen in standard laboratory assays, this species exhibited more robust rhythms under semi-natural conditions. We hypothesise that different ecological factors may have influenced these species to adopt different temporal niches.

A Subset of Circadian Neurons Expressing dTRPA1 Enables Appropriate Phasing of Activity Rhythms in Drosophila melanogaster Under Warm Temperatures.

Under conditions of prolonged durations of warmth, flies counter potential temperature stress by shifting their locomotor activity from day into night when the conditions are likely to be less harsh. Modulation of a rhythmic behavior such as this in response to the environment would require interaction between at least 2 neuronal systems: (1) a sensory system to receive input from the environment, and (2) the internal clock to correctly time rhythmic activity in response to this thermosensory input. Our previous studies found that a thermosensory mutant of the ion channel Drosophila Transient Receptor Potential-A1 (dTRPA1) failed to shift activity into the dark like control flies do and also identified the role of a specific cluster of the dTRPA1-expressing neurons, the dTRPA1sh+neurons necessary for this. In this study, we extended our previous findings and characterized the identity of these dTRPA1sh+ neurons based on their overlap with circadian neurons. Utilizing various genetic manipulations, we asked whether the overlapping neurons could be potential points of intersection between the 2 circuits that modulate behavior under warm temperature, meaning whether they function as both-sensory and clock neurons. We found that the molecular clock within the dTRPA1sh+ cluster was not necessary, but the expression of dTRPA1 in a subset of circadian neurons, the small ventrolateral neurons (sLNvs), was necessary in modulating phasing of behavior under warm temperature. Furthermore, attempting to identify the neuronal circuit, we were able to uncover the potential roles of serotonin and acetylcholine in modulating this temperature-dependent behavior. Finally, we also discuss possible parallel neuronal pathways that may exist to give rise to this modulation of behavior under warm temperature, thereby supporting and expanding the knowledge of the field about circuits that control temperature-mediated behavioral outcomes.

VANESSA-Shiny Apps for Accelerated Time-series Analysis and Visualization of Drosophila Circadian Rhythm and Sleep Data.

Chronobiologists and sleep researchers often need to estimate various rhythm and sleep parameters from locomotor activity data from different organisms. The available open-source or expensive paid tools do not offer consolidated analysis and visualization options in one bundle, are often cumbersome for users unfamiliar with coding, offer very low customization options, introduce sources of human errors by requiring users to manually pick period and power values from periodogram plots, and do not generate reproducible reports. We present VANESSA, a family of cross-platform apps written in R, which, in our opinion, have several advantages compared with available tools-(a) open-source; (b) automatic period-power detection; (c) time-series filtering and smoothing; (d) high-resolution publication-quality figures with dynamic coloring, resizing, and light/dark shading; (e) reproducible code-report generation; (f) analysis and visualization of multiple monitor files, defining genotypes and replicates separately; and (g) sleep profile analysis, various sleep parameter estimations, quantification, bout analysis, and latency analysis. The current version of the app is for data acquired through Drosophila Activity Monitors (DAM, TriKinetics) but can be easily extended to that from other data acquisition systems and from other organisms. We will continue to develop VANESSA with more useful features and version control will be done via archiving versions with significant changes on GitHub (https://github.com/orijitghosh/VANESSA-DAM) and Zenodo.

Drosophila Populations Reared Under Tropical Semi-natural Conditions Evolve Season-dependent Differences in Timing of Eclosion.

Circadian clocks are considered an evolutionary adaptation to environmental cycles, helping organisms to adapt to daily and seasonal changes. However, most studies on the evolution of circadian rhythms have been carried out in controlled laboratory conditions; hence evolution of circadian clocks and rhythms in organisms reared under the influence of naturally varying time cues is not well understood. To address this, we reared large outbred fly populations in an outdoor enclosure on our institutional grounds in Bengaluru, southern India for about 150 generations, at the same time maintaining their ancestral control populations under standard laboratory conditions. Studying their rhythms in eclosion, a vital behavior for Drosophila, in the laboratory and semi-natural environments revealed that flies reared under semi-natural conditions differed in the timing of eclosion under semi-natural conditions in a season-dependent manner from their laboratory-reared counterparts. These differences were manifested under harsh semi-natural environments but not under mild ones or in standard laboratory conditions. Further analysis revealed that this phenotype might be responsive to seasonal changes in temperature cycles which was confirmed in the laboratory with simulated light and temperature cycles that approximated semi-natural conditions. Our results highlight key intricacies on the relative impact of intensity and timing of environmental cues for predicting the timing of Drosophila eclosion under tropical naturalistic conditions. Overall, our research uncovers previously unexplored aspects of adaptive circadian timekeeping in complex natural conditions, offering valuable insight into the evolution of clocks.

A new player in circadian networks: Role of electrical synapses in regulating functions of the circadian clock.

Several studies have indicated that coherent circadian rhythms in behaviour can be manifested only when the underlying circadian oscillators function as a well-coupled network. The current literature suggests that circadian pacemaker neuronal networks rely heavily on communication mediated by chemical synapses comprising neuropeptides and neurotransmitters to regulate several behaviours and physiological processes. It has become increasingly clear that chemical synapses closely interact with electrical synapses and function together in the neuronal networks of most organisms. However, there are only a few studies which have examined the role of electrical synapses in circadian networks and here, we review our current understanding of gap junction proteins in circadian networks of various model systems. We describe the general mechanisms by which electrical synapses function in neural networks, their interactions with chemical neuromodulators and their contributions to the regulation of circadian rhythms. We also discuss the various methods available to characterize functional electrical synapses in these networks and the potential directions that remain to be explored to understand the roles of this relatively understudied mechanism of communication in modulating circadian behaviour.

Hsp40 overexpression in pacemaker neurons delays circadian dysfunction in a Drosophila model of Huntington's disease.

Circadian disturbances are early features of neurodegenerative diseases, including Huntington's disease (HD). Emerging evidence suggests that circadian decline feeds into neurodegenerative symptoms, exacerbating them. Therefore, we asked whether known neurotoxic modifiers can suppress circadian dysfunction. We performed a screen of neurotoxicity-modifier genes to suppress circadian behavioural arrhythmicity in a Drosophila circadian HD model. The molecular chaperones Hsp40 and HSP70 emerged as significant suppressors in the circadian context, with Hsp40 being the more potent mitigator. Upon Hsp40 overexpression in the Drosophila circadian ventrolateral neurons (LNv), the behavioural rescue was associated with neuronal rescue of loss of circadian proteins from small LNv soma. Specifically, there was a restoration of the molecular clock protein Period and its oscillations in young flies and a long-lasting rescue of the output neuropeptide Pigment dispersing factor. Significantly, there was a reduction in the expanded Huntingtin inclusion load, concomitant with the appearance of a spot-like Huntingtin form. Thus, we provide evidence implicating the neuroprotective chaperone Hsp40 in circadian rehabilitation. The involvement of molecular chaperones in circadian maintenance has broader therapeutic implications for neurodegenerative diseases. This article has an associated First Person interview with the first author of the paper.

Under warm ambient conditions, Drosophila melanogaster suppresses nighttime activity via the neuropeptide pigment dispersing factor.

Rhythmic locomotor behaviour of flies is controlled by an endogenous time-keeping mechanism, the circadian clock, and is influenced by environmental temperatures. Flies inherently prefer cool temperatures around 25°C, and under such conditions, time their locomotor activity to occur at dawn and dusk. Under relatively warmer conditions such as 30°C, flies shift their activity into the night, advancing their morning activity bout into the early morning, before lights-ON, and delaying their evening activity into early night. The molecular basis for such temperature-dependent behavioural modulation has been associated with core circadian clock genes, but the neuronal basis is not yet clear. Under relatively cool temperatures such as 25°C, the role of the circadian pacemaker ventrolateral neurons (LNvs), along with a major neuropeptide secreted by them, pigment dispersing factor (PDF), has been showed in regulating various aspects of locomotor activity rhythms. However, the role of the LNvs and PDF in warm temperature-mediated behavioural modulation has not been explored. We show here that flies lacking proper PDF signalling or the LNvs altogether, cannot suppress their locomotor activity resulting in loss of sleep during the middle of the night, and thus describe a novel role for PDF signalling and the LNvs in behavioural modulation under warm ambient conditions. In a rapidly warming world, such behavioural plasticity may enable organisms to respond to harsh temperatures in the environment.

Effects of Food Availability Cycles on Phase and Period of Activity-rest Rhythm in Drosophila melanogaster.

Foraging and feeding are indispensable for survival and their timing depends not only on the metabolic state of the animal but also on the availability of food resources in their environment. Since both these aspects are subject to change over time, these behaviors exhibit rhythmicity in occurrence. As the locomotor activity of an organism is related to its disposition to acquire food, and peak feeding in fruit flies has been shown to occur at a particular time of the day, we asked if cyclic food availability can entrain their rhythmic activity. By subjecting flies to cyclic food availability, that is, feeding-starvation (FS) cycles, we provided food cues contrasting to the preferred activity times and observed if this imposed cycling in food availability could entrain the activity-rest rhythm. We found that phase control, which is a property integral to entrainment, was not achieved despite increasing starvation duration of FS cycles (FS 12:12, FS 10:14, and FS 8:16). We also found that flies subjected to T21 and T26 FS cycles were unable to match period of the activity rhythm to short or long T-cycles. Taken together, these results show that external food availability cycles do not entrain the activity-rest rhythm of fruit flies. However, we find that starvation-induced hyperactivity causes masking which results in phase changes. In addition, T-cycle experiments resulted in minor period changes during FS treatment. These findings highlight that food cyclicity by itself may not be a potent zeitgeber but may act in unison with other abiotic factors like light and temperature to help flies time their activity appropriately.

Glutamatergic Synapse Dysfunction in Drosophila Neuromuscular Junctions Can Be Rescued by Proteostasis Modulation.

Glutamate is the major excitatory neurotransmitter in the nervous system, and the Drosophila glutamatergic neuromuscular junctions (NMJs) offer a tractable platform to understand excitatory synapse biology both in health and disease. Synaptopathies are neurodegenerative diseases that are associated with synaptic dysfunction and often display compromised proteostasis. One such rare, progressive neurodegenerative condition, Spinocerebellar Ataxia Type 3 (SCA3) or Machado-Joseph Disease (MJD), is characterized by cerebellar ataxia, Parkinsonism, and degeneration of motor neuron synapses. While the polyQ repeat mutant protein ataxin-3 is implicated in MJD, it is unclear how it leads to impaired synaptic function. In this study, we indicated that a Drosophila model of MJD recapitulates characteristics of neurodegenerative disorders marked by motor neuron dysfunction. Expression of 78 polyQ repeats of mutant ataxin-3 protein in Drosophila motor neurons resulted in behavioral defects, such as impaired locomotion in both larval and adult stages. Furthermore, defects in eclosion and lifespan were observed in adult flies. Detailed characterization of larval glutamatergic neuromuscular junctions (NMJs) revealed defects in morphological features along with compromised NMJ functioning. Autophagy, one of the key proteostasis pathways, is known to be impaired in the case of several synaptopathies. Our study reveals that overexpression of the autophagy-related protein Atg8a rescued behavioral defects. Thus, we present a model for glutamatergic synapse dysfunction that recapitulates synaptic and behavioral deficits and show that it is an amenable system for carrying out genetic and chemical biology screens to identify potential therapeutic targets for synaptopathies.