TDP-43 impairs sleep in Drosophila through Ataxin-2-dependent metabolic disturbance.

Neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia are associated with substantial sleep disruption, which may accelerate cognitive decline and brain degeneration. Here, we define a role for trans-activation response element (TAR) DNA binding protein 43 (TDP-43), a protein associated with human neurodegenerative disease, in regulating sleep using Drosophila. Expression of TDP-43 severely disrupts sleep, and the sleep deficit is rescued by Atx2 knockdown. Brain RNA sequencing revealed that Atx2 RNA interference regulates transcripts enriched for small-molecule metabolic signaling in TDP-43 brains. Focusing on these Atx2-regulated genes, we identified suppressors of the TDP-43 sleep phenotype enriched for metabolism pathways. Knockdown of Atx2 or treatment with rapamycin attenuated the sleep phenotype and mitigated the disruption of small-molecule glycogen metabolism caused by TDP-43. Our findings provide a connection between toxicity of TDP-43 and sleep disturbances and highlight key aspects of metabolism that interplay with TDP-43 toxicity upon Atx2 rescue.

Energetic Demands Regulate Sleep-Wake Rhythm Circuit Development.

Sleep and feeding patterns lack a clear daily rhythm during early life. As diurnal animals mature, feeding is consolidated to the day and sleep to the night. Circadian sleep patterns begin with formation of a circuit connecting the central clock to arousal output neurons; emergence of circadian sleep also enables long-term memory (LTM). However, the cues that trigger the development of this clock-arousal circuit are unknown. Here, we identify a role for nutritional status in driving sleep-wake rhythm development in Drosophila larvae. We find that in the 2nd instar (L2) period, sleep and feeding are spread across the day; these behaviors become organized into daily patterns by L3. Forcing mature (L3) animals to adopt immature (L2) feeding strategies disrupts sleep-wake rhythms and the ability to exhibit LTM. In addition, the development of the clock (DN1a)-arousal (Dh44) circuit itself is influenced by the larval nutritional environment. Finally, we demonstrate that larval arousal Dh44 neurons act through glucose metabolic genes to drive onset of daily sleep-wake rhythms. Together, our data suggest that changes to energetic demands in developing organisms triggers the formation of sleep-circadian circuits and behaviors.

Stem cell-specific ecdysone signaling regulates the development and function of a Drosophila sleep homeostat.

Complex behaviors arise from neural circuits that are assembled from diverse cell types. Sleep is a conserved and essential behavior, yet little is known regarding how the nervous system generates neuron types of the sleep-wake circuit. Here, we focus on the specification of Drosophila sleep-promoting neurons-long-field tangential input neurons that project to the dorsal layers of the fan-shaped body neuropil in the central complex (CX). We use lineage analysis and genetic birth dating to identify two bilateral Type II neural stem cells that generate these dorsal fan-shaped body (dFB) neurons. We show that adult dFB neurons express Ecdysone-induced protein E93, and loss of Ecdysone signaling or E93 in Type II NSCs results in the misspecification of the adult dFB neurons. Finally, we show that E93 knockdown in Type II NSCs affects adult sleep behavior. Our results provide insight into how extrinsic hormonal signaling acts on NSCs to generate neuronal diversity required for adult sleep behavior. These findings suggest that some adult sleep disorders might derive from defects in stem cell-specific temporal neurodevelopmental programs.

Developmental emergence of sleep rhythms enables long-term memory in Drosophila.

In adulthood, sleep-wake rhythms are one of the most prominent behaviors under circadian control. However, during early life, sleep is spread across the 24-hour day. The mechanism through which sleep rhythms emerge, and consequent advantage conferred to a juvenile animal, is unknown. In the second-instar Drosophila larvae (L2), like in human infants, sleep is not under circadian control. We identify the precise developmental time point when the clock begins to regulate sleep in Drosophila, leading to emergence of sleep rhythms in early third-instars (L3). At this stage, a cellular connection forms between DN1a clock neurons and arousal-promoting Dh44 neurons, bringing arousal under clock control to drive emergence of circadian sleep. Last, we demonstrate that L3 but not L2 larvae exhibit long-term memory (LTM) of aversive cues and that this LTM depends upon deep sleep generated once sleep rhythms begin. We propose that the developmental emergence of circadian sleep enables more complex cognitive processes, including the onset of enduring memories.

Neurofibromin 1 regulates early developmental sleep in Drosophila.

Sleep disturbances are common in neurodevelopmental disorders, but knowledge of molecular factors that govern sleep in young animals is lacking. Evidence across species, including Drosophila, suggests that juvenile sleep has distinct functions and regulatory mechanisms in comparison to sleep in maturity. In flies, manipulation of most known adult sleep regulatory genes is not associated with sleep phenotypes during early developmental (larval) stages. Here, we examine the role of the neurodevelopmental disorder-associated gene Neurofibromin 1 (Nf1) in sleep during numerous developmental periods. Mutations in Neurofibromin 1 (Nf1) are associated with sleep and circadian disorders in humans and adult flies. We find in flies that Nf1 acts to regulate sleep across the lifespan, beginning during larval stages. Nf1 is required in neurons for this function, as is signaling via the Alk pathway. These findings identify Nf1 as one of a small number of genes positioned to regulate sleep across developmental periods.

Intrinsic maturation of sleep output neurons regulates sleep ontogeny in Drosophila.

The maturation of sleep behavior across a lifespan (sleep ontogeny) is an evolutionarily conserved phenomenon. Mammalian studies have shown that in addition to increased sleep duration, early life sleep exhibits stark differences compared with mature sleep with regard to sleep states. How the intrinsic maturation of sleep output circuits contributes to sleep ontogeny is poorly understood. The fruit fly Drosophila melanogaster exhibits multifaceted changes to sleep from juvenile to mature adulthood. Here, we use a non-invasive probabilistic approach to investigate the changes in sleep architecture in juvenile and mature flies. Increased sleep in juvenile flies is driven primarily by a decreased probability of transitioning to wake and characterized by more time in deeper sleep states. Functional manipulations of sleep-promoting neurons in the dorsal fan-shaped body (dFB) suggest that these neurons differentially regulate sleep in juvenile and mature flies. Transcriptomic analysis of dFB neurons at different ages and a subsequent RNAi screen implicate the genes involved in dFB sleep circuit maturation. These results reveal that the dynamic transcriptional states of sleep output neurons contribute to the changes in sleep across the lifespan.

Synaptic dysfunction connects autism spectrum disorder and sleep disturbances: A perspective from studies in model organisms.

Sleep disturbances (SD) accompany many neurodevelopmental disorders, suggesting SD is a transdiagnostic process that can account for behavioral deficits and influence underlying neuropathogenesis. Autism Spectrum Disorder (ASD) comprises a complex set of neurodevelopmental conditions characterized by challenges in social interaction, communication, and restricted, repetitive behaviors. Diagnosis of ASD is based primarily on behavioral criteria, and there are no drugs that target core symptoms. Among the co-occurring conditions associated with ASD, SD are one of the most prevalent. SD often arises before the onset of other ASD symptoms. Sleep interventions improve not only sleep but also daytime behaviors in children with ASD. Here, we examine sleep phenotypes in multiple model systems relevant to ASD, e.g., mice, zebrafish, fruit flies and worms. Given the functions of sleep in promoting brain connectivity, neural plasticity, emotional regulation and social behavior, all of which are of critical importance in ASD pathogenesis, we propose that synaptic dysfunction is a major mechanism that connects ASD and SD. Common molecular targets in this interplay that are involved in synaptic function might be a novel avenue for therapy of individuals with ASD experiencing SD. Such therapy would be expected to improve not only sleep but also other ASD symptoms.

Getting into rhythm: developmental emergence of circadian clocks and behaviors.

Circadian clocks keep time to coordinate diverse behaviors and physiological functions. While molecular circadian rhythms are evident during early development, most behavioral rhythms, such as sleep-wake, do not emerge until far later. Here, we examine the development of circadian clocks, outputs, and behaviors across phylogeny, with a particular focus on Drosophila. We explore potential mechanisms for how central clocks and circadian output loci establish communication, and discuss why from an evolutionary perspective sleep-wake and other behavioral rhythms emerge long after central clocks begin keeping time.

Treatment of Insomnia with Zaleplon in HIV+ Significantly Improves Sleep and Depression.

More than 50% of individuals who are HIV positive report insomnia, which can reduce HIV treatment adherence, impair quality of life, and contribute to metabolic dysfunction. Major depressive disorder is also highly comorbid in this population, leading to further impairment. There is evidence that treating insomnia may improve not only sleep, but depression and metabolic function, as well. The present study aimed to examine the effects of pharmacotherapeutic treatment of insomnia on sleep, depression, and metabolic functioning in individuals with HIV. 20 individuals with asymptomatic seropositive HIV and comorbid insomnia and depression were administered zaleplon for 6 weeks. Insomnia severity was assessed using the Insomnia Severity Index and Epworth Sleepiness Scale, and depression severity was assessed using the Quick Inventory of Depression, both prior to treatment and 6 weeks post treatment. Metabolomic changes were assessed using a comprehensive platform measuring ~2000 lipid features and polar metabolites. Linear mixed effects models demonstrated that 6 weeks of treatment with zaleplon significantly improved symptoms of both insomnia and depression. Metabolomic analyses also demonstrated that changes in insomnia severity were associated with significant changes in key branched chain amino acid metabolites. Our results show that improvement in insomnia is associated with reduced depressive symptoms and beneficial metabolomic changes. Additionally, changes in key branched chain amino acid metabolites following treatment may serve as useful biomarkers of treatment response.

Randomized Noninferiority Trial of Telehealth Delivery of Cognitive Behavioral Treatment of Insomnia Compared to In-Person Care.

Objective: Insomnia is prevalent and is associated with a range of negative sequelae. Cognitive behavioral treatment for insomnia (CBT-I) is the recommended intervention, but availability is limited. Telehealth provides increased access, but its efficacy is not certain. The objective of this study was to compare the efficacy of CBT-I delivered by telehealth to in-person treatment and to a waitlist control.Methods: Individuals with DSM-5 insomnia disorder (n = 60) were randomized to telehealth CBT-I, in-person CBT-I, or 8-week waitlist control. CBT-I was delivered over 6-8 weekly sessions by video telehealth or in-person in an outpatient clinic. Follow-up assessments were at 2 weeks and 3 months posttreatment. The Insomnia Severity Index (ISI) was the primary outcome. Change in ISI score was compared between the CBT-I group in an intent-to-treat, noninferiority analysis using an a priori margin of -3.0 points. All analyses were conducted using mixed-effects models. Data collection occurred from November 2017-July 2020.Results: The mean (SD) change in ISI score from baseline to 3-month follow-up was -7.8 (6.1) points for in-person CBT-I, -7.5 (6.9) points for telehealth, and -1.6 (2.1) for waitlist, and the difference between the CBT-I groups was not statistically significant (t28 = -0.98, P = .33). The lower confidence limit of this between-group difference in the mean ISI changes was greater than the a priori margin of -3.0 points, indicating that telehealth treatment was not inferior to in-person treatment. There were significant improvements on most secondary outcome measures but no group differences.Conclusions: Telehealth CBT-I may produce clinically significant improvements in insomnia severity that are noninferior to in-person treatment. CBT-I is also associated with significant gains across a range of domains of functioning. Telehealth is a promising option for increasing access to treatment without loss of clinical gains.Trial Registration: ClinicalTrials.gov identifier: NCT03328585.

The CHD8/CHD7/Kismet family links blood-brain barrier glia and serotonin to ASD-associated sleep defects.

Sleep disturbances in autism and neurodevelopmental disorders are common and adversely affect patient's quality of life, yet the underlying mechanisms are understudied. We found that individuals with mutations in CHD8, among the highest-confidence autism risk genes, or CHD7 suffer from disturbed sleep maintenance. These defects are recapitulated in Drosophila mutants affecting kismet, the sole CHD8/CHD7 ortholog. We show that Kismet is required in glia for early developmental and adult sleep architecture. This role localizes to subperineurial glia constituting the blood-brain barrier. We demonstrate that Kismet-related sleep disturbances are caused by high serotonin during development, paralleling a well-established but genetically unsolved autism endophenotype. Despite their developmental origin, Kismet's sleep architecture defects can be reversed in adulthood by a behavioral regime resembling human sleep restriction therapy. Our findings provide fundamental insights into glial regulation of sleep and propose a causal mechanistic link between the CHD8/CHD7/Kismet family, developmental hyperserotonemia, and autism-associated sleep disturbances.

Pathogenic variants in SMARCA5, a chromatin remodeler, cause a range of syndromic neurodevelopmental features.

Intellectual disability encompasses a wide spectrum of neurodevelopmental disorders, with many linked genetic loci. However, the underlying molecular mechanism for more than 50% of the patients remains elusive. We describe pathogenic variants in SMARCA5, encoding the ATPase motor of the ISWI chromatin remodeler, as a cause of a previously unidentified neurodevelopmental disorder, identifying 12 individuals with de novo or dominantly segregating rare heterozygous variants. Accompanying phenotypes include mild developmental delay, frequent postnatal short stature and microcephaly, and recurrent dysmorphic features. Loss of function of the SMARCA5 Drosophila ortholog Iswi led to smaller body size, reduced sensory dendrite complexity, and tiling defects in larvae. In adult flies, Iswi neural knockdown caused decreased brain size, aberrant mushroom body morphology, and abnormal locomotor function. Iswi loss of function was rescued by wild-type but not mutant SMARCA5. Our results demonstrate that SMARCA5 pathogenic variants cause a neurodevelopmental syndrome with mild facial dysmorphia.

The chromatin remodeler ISWI acts during Drosophila development to regulate adult sleep.

Sleep disruptions are among the most commonly reported symptoms across neurodevelopmental disorders (NDDs), but mechanisms linking brain development to normal sleep are largely unknown. From a Drosophila screen of human NDD-associated risk genes, we identified the chromatin remodeler Imitation SWItch/SNF (ISWI) to be required for adult fly sleep. Loss of ISWI also results in disrupted circadian rhythms, memory, and social behavior, but ISWI acts in different cells and during distinct developmental times to affect each of these adult behaviors. Specifically, ISWI expression in type I neuroblasts is required for both adult sleep and formation of a learning-associated brain region. Expression in flies of the human ISWI homologs SMARCA1 and SMARCA5 differentially rescues adult phenotypes, while de novo SMARCA5 patient variants fail to rescue sleep. We propose that sleep deficits are a primary phenotype of early developmental origin in NDDs and point toward chromatin remodeling machinery as critical for sleep circuit formation.

A Drosophila model of sleep restriction therapy for insomnia.

Insomnia is the most common sleep disorder among adults, especially affecting individuals of advanced age or with neurodegenerative disease. Insomnia is also a common comorbidity across psychiatric disorders. Cognitive behavioral therapy for insomnia (CBT-I) is the first-line treatment for insomnia; a key component of this intervention is restriction of sleep opportunity, which optimizes matching of sleep ability and opportunity, leading to enhanced sleep drive. Despite the well-documented efficacy of CBT-I, little is known regarding how CBT-I works at a cellular and molecular level to improve sleep, due in large part to an absence of experimentally-tractable animals models of this intervention. Here, guided by human behavioral sleep therapies, we developed a Drosophila model for sleep restriction therapy (SRT) of insomnia. We demonstrate that restriction of sleep opportunity through manipulation of environmental cues improves sleep efficiency in multiple short-sleeping Drosophila mutants. The response to sleep opportunity restriction requires ongoing environmental inputs, but is independent of the molecular circadian clock. We apply this sleep opportunity restriction paradigm to aging and Alzheimer's disease fly models, and find that sleep impairments in these models are reversible with sleep restriction, with associated improvement in reproductive fitness and extended lifespan. This work establishes a model to investigate the neurobiological basis of CBT-I, and provides a platform that can be exploited toward novel treatment targets for insomnia.

Social Behavioral Deficits with Loss of Neurofibromin Emerge from Peripheral Chemosensory Neuron Dysfunction.

Neurofibromatosis type 1 (NF1) is a neurodevelopmental disorder associated with social and communicative disabilities. The cellular and circuit mechanisms by which loss of neurofibromin 1 (Nf1) results in social deficits are unknown. Here, we identify social behavioral dysregulation with Nf1 loss in Drosophila. These deficits map to primary dysfunction of a group of peripheral sensory neurons. Nf1 regulation of Ras signaling in adult ppk23+ chemosensory cells is required for normal social behaviors in flies. Loss of Nf1 attenuates ppk23+ neuronal activity in response to pheromones, and circuit-specific manipulation of Nf1 expression or neuronal activity in ppk23+ neurons rescues social deficits. This disrupted sensory processing gives rise to persistent changes in behavior beyond the social interaction, indicating a sustained effect of an acute sensory misperception. Together our data identify a specific circuit mechanism through which Nf1 regulates social behaviors and suggest social deficits in NF1 arise from propagation of sensory misinformation.

Identification of a molecular basis for the juvenile sleep state.

Across species, sleep in young animals is critical for normal brain maturation. The molecular determinants of early life sleep remain unknown. Through an RNAi-based screen, we identified a gene, pdm3, required for sleep maturation in Drosophila. Pdm3, a transcription factor, coordinates an early developmental program that prepares the brain to later execute high levels of juvenile adult sleep. PDM3 controls the wiring of wake-promoting dopaminergic (DA) neurites to a sleep-promoting region, and loss of PDM3 prematurely increases DA inhibition of the sleep center, abolishing the juvenile sleep state. RNA-Seq/ChIP-Seq and a subsequent modifier screen reveal that pdm3 represses expression of the synaptogenesis gene Msp300 to establish the appropriate window for DA innervation. These studies define the molecular cues governing sleep behavioral and circuit development, and suggest sleep disorders may be of neurodevelopmental origin.

Sleep: The Balm of Hurt Minds.

Two new studies use Drosophila to unravel the role of sleep in clearance of damaged neural processes.

Precision Medicine for Insomnia.

Insomnia affects up to 15% of the US population. There are effective pharmacologic and behavioral treatments for insomnia; however, there is often no one-size-fits-all intervention. This article discusses the leading behavioral treatment of insomnia, cognitive behavioral therapy for insomnia, and its ability to be tailored to an individual's specific symptoms. It then discusses pharmacologic options for treating insomnia, and offers some guidance on medication selection to enhance personalized care. In addition, it discusses how the current evidence base can help providers make choices between pharmacologic and behavioral treatments.

Quantitative imaging of sleep behavior in Caenorhabditis elegans and larval Drosophila melanogaster.

Sleep is nearly universal among animals, yet remains poorly understood. Recent work has leveraged simple model organisms, such as Caenorhabditis elegans and Drosophila melanogaster larvae, to investigate the genetic and neural bases of sleep. However, manual methods of recording sleep behavior in these systems are labor intensive and low in throughput. To address these limitations, we developed methods for quantitative imaging of individual animals cultivated in custom microfabricated multiwell substrates, and used them to elucidate molecular mechanisms underlying sleep. Here, we describe the steps necessary to design, produce, and image these plates, as well as analyze the resulting behavioral data. We also describe approaches for experimentally manipulating sleep. Following these procedures, after ~2 h of experimental preparation, we are able to simultaneously image 24 C. elegans from the second larval stage to adult stages or 20 Drosophila larvae during the second instar life stage at a spatial resolution of 10 or 27 µm, respectively. Although this system has been optimized to measure activity and quiescence in Caenorhabditis larvae and adults and in Drosophila larvae, it can also be used to assess other behaviors over short or long periods. Moreover, with minor modifications, it can be adapted for the behavioral monitoring of a wide range of small animals.

Starvation resistance is associated with developmentally specified changes in sleep, feeding and metabolic rate.

Food shortage represents a primary challenge to survival, and animals have adapted diverse developmental, physiological and behavioral strategies to survive when food becomes unavailable. Starvation resistance is strongly influenced by ecological and evolutionary history, yet the genetic basis for the evolution of starvation resistance remains poorly understood. The fruit fly Drosophila melanogaster provides a powerful model for leveraging experimental evolution to investigate traits associated with starvation resistance. While control populations only live a few days without food, selection for starvation resistance results in populations that can survive weeks. We have previously shown that selection for starvation resistance results in increased sleep and reduced feeding in adult flies. Here, we investigate the ontogeny of starvation resistance-associated behavioral and metabolic phenotypes in these experimentally selected flies. We found that selection for starvation resistance resulted in delayed development and a reduction in metabolic rate in larvae that persisted into adulthood, suggesting that these traits may allow for the accumulation of energy stores and an increase in body size within these selected populations. In addition, we found that larval sleep was largely unaffected by starvation selection and that feeding increased during the late larval stages, suggesting that experimental evolution for starvation resistance produces developmentally specified changes in behavioral regulation. Together, these findings reveal a critical role for development in the evolution of starvation resistance and indicate that selection can selectively influence behavior during defined developmental time points.