David A. Prober - US grants

Greater thari 10% of hurhahs suffer chronic sleep disorders, hut the genetic m[unreadable]:hanisims that regulate sleep are largely unknown. The identification of defective Hypocretin/Orexin i[Hcrt) signaling as a cause of narcolepsy provided ia genetic enUry point into sleepresearch.but an effective treatment for this disorder has not yet been found. Moreover, only a sniall fraction of sleep disorders are associated with narcolepsy, indicating that additional genes that control sleep and wakefulness remain to be identified. The objective of this proposal is to use zebrafish as a simpile and cost-effective vertebrate rnodel system to study the genetics of Hcrt Jsighaling andsleep. Zebrafish are an excellent model for these studies because they are amenable to high-throughput genetic screens and, unlike invertebra:tes, have a Hcrt ortholog and the basic brain stl-iictures that regulate sleepin mammals. I have shown that Hcrt overexpression causes an insomhia-like phenotype in zebrafish larvae. In Specific Aim 1,1 will use pharmacological agents to determine the genetic and neurologic mechanisms by which Hcrt dverexpress;ion induces this phenotype^ These experiments vvill use reagents identified in a small molecule screen that I performed during the K99 phase of this grant. The results of these experiments will improve understanding Of Hcrt function and may provide cIue:S to the basis of chronic insomnia. In Specific Aim 2,1 will study secreted peptides that caused specific sleep phenotypes in the genetic overexpression screen that I performed during the K99 phase of this grant. These experiments vyill confirm results frorh the screen and characterize potentially novel genetic mechanisms that regulate sleep. My long-term career goalis to elucidate: the genetic and neurologic mechanisms that regulate sleep/wake states, with the hope that this knowledge will lead to treatments for sleep disorders. Caltech is an excellent environnient to pursue this goal since there are several labs performing outstanding research in the genetics of behavior arid behavioral neuroscience, as well as labs studying the regulation of sleep in other model systems.

DESCRIPTION (provided by applicant): Over 10% of Americans suffer from chronic sleep disorders, with an estimated annual cost of $100 billion. Drug and alcohol addiction severely disrupt sleep, and sleep disorders increase the risk of addiction and relapse. Understanding the mechanisms that regulate sleep is thus critical for preventing and treating addiction. Furthermore, most drugs used to treat insomnia, the most common sleep disorder, act by inhibiting GABA receptors, which are relatively non-specific targets for sleep. These drugs only ameliorate some symptoms and are often addictive. Thus, new drugs that target more specific sleep regulators are needed. We recently established zebrafish as a vertebrate system for studying the genetic and neural mechanisms that regulate sleep. Zebrafish are a useful model for these studies because they have the basic brain structures and genes that are thought to regulate mammalian sleep, but are also optically transparent and amenable to high- throughput screens and behavior assays. This proposal has four specific aims. First, we will use genetic and pharmacological approaches to determine which adenosine receptors regulate zebrafish sleep/wake states. Second, we will determine which neural substrates are used by adenosine to regulate zebrafish sleep. Third, we will test the hypothesis that adenosine promotes sleep by inhibiting the Hypocretin system. Fourth, we will test the hypothesis that adenosine regulates sleep by modulating the activities of other sleep regulatory systems. These experiments will clarify how adenosine regulates sleep, which may provide clues to the basis of sleep disorders and suggest novel therapies for sleep disorders and addiction.

DESCRIPTION (provided by applicant): Over 10% of humans suffer chronic sleep disorders, but the genetic and neural mechanisms that regulate sleep are poorly understood. The identification of defective Hypocretin/Orexin (Hcrt) signaling as a cause of narcolepsy provided a genetic entry point into sleep research, but an effective treatment for this disorder has not yet been found. Moreover, only a small fraction of sleep disorders are associated with narcolepsy, indicating that additional genes and neurons that control sleep and wakefulness remain to be identified. The objective of this proposal is to use zebrafish as a simple and cost-effective vertebrate model system to study Hcrt signaling and sleep. Zebrafish are a useful model for these studies because they have the basic brain structures and genes that are thought to regulate mammalian sleep, but are also optically transparent and amenable to high-throughput behavior assays. The proposed research has three aims. First, we will characterize the development of larval zebrafish Hcrt neurons at the single neuron level and test hypotheses about their development. Second, we will use genetic and pharmacological approaches to determine which neurons are activated by Hcrt signaling and which neurotransmitter systems are required for Hcrt-induced wakefulness. Third, we will determine whether other sleep regulators, including adenosine, melatonin, and Kv3-type potassium channels, affect sleep via the Hcrt system. These experiments will improve our understanding of Hcrt neuron development and function, may provide clues to the basis of sleep disorders such as chronic insomnia, and may lead to novel therapies for these disorders. PUBLIC HEALTH RELEVANCE: Over 10% of humans suffer chronic sleep disorders, but the causes of most of these disorders are unknown. This proposal will use zebrafish to examine how sleep is regulated by studying a gene whose loss causes the human sleep disorder narcolepsy and may be involved in other sleep disorders. The proposed studies will improve understanding of the genetic and neuronal mechanisms that regulate sleep and may suggest new strategies to treat sleep disorders.

Sleep and wake states are regulated by several brain regions that act via multiple neurotransmitters, neuromodulators and neuropeptides. While several such brain regions and neuropeptides have been identified, additional mechanisms that regulate sleep likely remain to be discovered. Indeed, we recently identified a family of FMRFamide-like neuropeptides that are sufficient to induce a sleep-like state in C. elegans, and found that the zebrafish FMRFamide neuropeptide NPVF is sufficient to induce sleep-like states in both C. elegans and zebrafish. These observations suggest that NPVF acts in an ancient and central mechanism that regulates sleep. While rodents are commonly used for vertebrate sleep studies, genetic tools needed to study NPVF in rodents have not been described. The zebrafish is an alternative vertebrate model that exhibits behavioral, anatomical, genetic and pharmacological conservation of mammalian sleep, and studies using this simple and inexpensive vertebrate model will provide a rationale to invest the significant time, labor and expense required to generate tools to study NPVF in mice. While the zebrafish has some limitations as a sleep model, its amenability to genetic, optogenetic and pharmacological approaches, as well as its transparency and relatively simple yet conserved vertebrate brain, provide advantages for sleep studies that we exploit in this proposal. Both the protein sequence and hypothalamic expression of NPVF are conserved in zebrafish, rodents and humans, suggesting that findings in zebrafish will be relevant to humans. In Specific Aim 1 we use gain- and loss-of-function genetics to determine whether one or more of the three mature peptides produced by the NPVF preproprotein are necessary and sufficient to promote sleep. We will also determine which receptors mediate the role of NPVF in sleep. In Specific Aim 2 we will test the hypothesis that the ~15 npvf-expressing neurons are necessary and sufficient to promote sleep. We will use noninvasive and high-throughput optogenetic and chemogenetic assays to stimulate, inhibit and ablate NPVF neurons in freely behaving larvae. In Specific Aim 3 we will identify neurons that are activated or inhibited in response to stimulation and inhibition of NPVF neurons using two-photon single plane illumination microscopy and whole-brain calcium imaging. The zebrafish is the only vertebrate model in which whole-brain calcium imaging is currently feasible. This approach will in an unbiased and comprehensive manner identify neurons that may mediate the effects of NPVF on sleep, which we will functionally test using genetics and optogenetics. This project will establish a novel and evolutionarily conserved sleep-regulatory system, and may eventually lead to new therapies for sleep disorders. Because abnormal sleep is associated with several neuropsychiatric disorders and may be causal in some cases, this project may also eventually lead to improved therapies for some forms of neuropsychiatric disorders.

Chronic sleep disorders are pervasive in society, have poor therapeutic options, and cause an estimated annual economic burden of $100 billion. Despite the impact of sleep disorders, the fact that we sleep for a third of our lives, and the evolutionary conservation of sleep-like states, mechanisms that regulate sleep remain poorly understood. Understanding these mechanisms is important because sleep performs an essential function and is necessary in order to eventually develop therapies for sleep disorders. Progress has been hindered in part by the complexity of mammalian brains and the challenge of performing screens in mammals. To overcome these limitations, we and others recently demonstrated behavioral, anatomical, genetic and pharmacological conservation of sleep between zebrafish and mammals, establishing zebrafish as a simple and inexpensive vertebrate model for sleep. We recently used zebrafish to perform the first large-scale screen for genes that regulate vertebrate sleep. We found that overexpression of the neuropeptide neuromedin U (Nmu) promotes locomotor activity and inhibits sleep. While one study showed that Nmu can transiently disrupt sleep in rats, its role in sleep has not been extensively studied, the mechanism through which it affects sleep is unclear, and a role for nmu-expressing neurons in sleep has not been explored. We will determine the genetic and neurological mechanisms through which Nmu and nmu-expressing neurons regulate sleep using approaches that exploit advantageous features of zebrafish. In Specific Aim 1 we will test the hypothesis that Nmu signaling is required for normal sleep/wake behaviors using nmu and nmu receptor mutants, and explore whether Nmu is required for arousal in specific contexts. We will also determine the genetic mechanisms through which Nmu affects sleep by testing whether zebrafish that lack neuromodulators and neuropeptides known to regulate sleep suppress Nmu gain- and loss-of-function phenotypes. These experiments will identify genetic mechanisms through which Nmu regulates sleep and place it in the context of established sleep pathways. In Specific Aim 2 we will test the hypothesis that nmu-expressing neurons inhibit sleep by using optogenetic and chemogenetic assays to stimulate, inhibit and ablate them, and assay effects on behavior. In Specific Aim 3 we will test the hypothesis that Nmu promotes wakefulness by stimulating corticotropin releasing hormone (crh)-expressing brainstem neurons, and that Crh signaling, the locus coeruleus and noradrenaline are required for Nmu-induced arousal. Validation of this hypothesis will identify a novel neural circuit that regulates arousal. Because disrupted sleep is associated with several neurological disorders and may be causal in some cases, this project may eventually lead to improved therapies for sleep disorders and some neurological disorders. Nmu signaling is a particularly attractive drug target because Nmu acts via G- protein coupled receptors, which are amenable to drug modulation, although much additional work will be needed before the basic research proposed here can be translated into therapies.

ABSTRACT Sleep is regulated by homeostatic and circadian mechanisms. Several factors mediate homeostatic control of sleep and mechanisms that regulate the circadian clock are well understood, but little is known about how the circadian clock regulates sleep. Melatonin (MT) is a good candidate link between the circadian clock and sleep because the circadian clock regulates its production, it can induce sleep in several diurnal species, including humans, and its loss is associated with reduced sleep in humans. MT may play an ancient role in regulating rest/activity states, as it does so in animals such as nematodes and zooplankton. However, the role of MT in vertebrate sleep is controversial for two reasons. First, most nocturnal lab mouse strains do not synthesize MT, yet they exhibit circadian control of sleep. However, MT synthesis peaks at night in both diurnal and nocturnal animals, and MT does not induce sleep in nocturnal animals, suggesting that MT only regulates sleep in diurnal animals. Second, a large number of studies in vertebrates have failed to show a consistent role for MT in sleep, suggesting that it is not a central sleep regulator, but rather has species-specific roles. However, these studies used pinealectomy (Px), a crude, imprecise and invasive procedure that does not address the function of MT in a clean manner. Thus, it is likely that distinct effects of Px result from differences in the Px procedure in different species and in different labs. The pineal gland also likely has functions in addition to MT production, so Px may cause effects that obscure MT-dependent phenotypes. These confounds are avoided by using genetics to create MT-deficient animals. We have addressed this question using zebrafish, a diurnal vertebrate that exhibits behavioral, anatomical, genetic and pharmacological conservation of mammalian sleep. We found that circadian regulation of sleep is abolished in zebrafish that lack MT due to mutation of arylalkylamine N-acetyltransferase (aanat2). This finding suggests that MT mediates the circadian regulation of sleep in a diurnal vertebrate and provides a basis to explore genetic and neurological mechanisms through which the circadian system and MT regulate sleep. In Specific Aim 1 we use genetics to determine which MT receptors are required for circadian regulation of sleep and for sleep induced by exogenous MT. In Specific Aim 2 we identify neurons that are activated or inhibited in response to exogenous MT and in MT-deficient animals using cfos in situ hybridization and whole-brain GCaMP6s calcium imaging. In Specific Aim 3 we use genetics and mass spectrometry to test the hypothesis that MT promotes sleep by stimulating adenosine signaling. If correct, this would suggest a simple mechanism that integrates homeostatic and circadian control of sleep. This project will help to reveal how MT, and thus the circadian clock, regulates sleep in a diurnal vertebrate. Sleep disorders are common, have poor therapeutic options and cause an annual economic burden of $100 billion. Our findings may eventually lead to improved therapies for sleep disorders that may also be useful to treat neuropsychiatric disorders that are exacerbated by disrupted sleep.

Light affects sleep indirectly by entraining the circadian clock, but also directly and rapidly via a phenomenon known as the masking or direct effect of light on behavior. Masking refers to the observation that light exposure at night induces sleep in nocturnal animals and suppresses sleep in diurnal animals, including humans, whereas dark exposure during the day inhibits sleep in nocturnal animals. While masking is widespread in the animal kingdom, the mechanisms that mediate masking are largely unknown. Understanding mechanisms that regulate sleep is important because over 10% of Americans suffer from chronic sleep disorders that have poor therapeutic options and cause an annual economic burden of $100 billion. Understanding how masking in particular affects sleep is important because at least some of the sleep disruption observed in modern societies results from masking due to widespread exposure to artificial light at night. Our preliminary experiments using zebrafish suggest that the neuropeptide prokineticin 2 (Prok2) regulates masking. To our knowledge, this is the first gene shown to regulate masking beyond the initial step of light detection by melanopsin in the retina, and thus it provides a foothold to explore the genetic and neurological mechanisms through which light directly affects sleep. Although a similar phenotype was observed in rodents, a role in masking was not explored, and the developmental defects of prok2 mutant mice confound sleep studies. The zebrafish is a vertebrate model that exhibits behavioral, anatomical, genetic and pharmacological conservation of mammalian sleep. While the zebrafish has some limitations as a sleep model, its amenability to genetic, optogenetic and pharmacological approaches, as well as its transparency and relatively simple yet conserved vertebrate brain, and the lack of developmental defects in prok2 mutant animals, provide advantages for sleep studies that we exploit in this proposal. The zebrafish is particularly appropriate for studying mechanisms that may underlie masking in humans because it is a diurnal vertebrate. In Specific Aim 1 we use gain- and loss-of-function genetics, as well as optogenetic and chemogenetic tools, to test the hypotheses that Prok2 and prok2-expressing neurons regulate masking. We also test whether our findings for Prok2 apply to other peptides that have been proposed to regulate behavior in a manner similar to Prok2. In Specific Aim 2, we explore potential mechanisms through which Prok2 regulates masking, such as by regulating the activity of the noradrenergic locus coeruleus (Aim 2A), the expression of the sleep-promoting neuropeptide galanin in the hypothalamus (Aim 2B) or other sleep regulatory pathways. These studies will for the first time identify genes and neurons in the brain that regulate masking, and thus provide a basis to study the genetic and neurological mechanisms that underlie this phenomenon. Because Prok2 acts via G-protein coupled receptors, which are amenable to small molecule modulation, this work may eventually lead to novel therapies for sleep disorders and for neuropsychiatric disorders that are exacerbated by disrupted sleep.

PROJECT SUMMARY It is widely thought that identifying how neurons are connected to each other in a brain circuit, its wiring diagram, is a necessary step towards understanding how brain activity gives rise to behavior, and how it is perturbed by disease. Unfortunately, currently available methods have limitations that make it challenging to visualize these brain wiring diagrams. In addition, there is an urgent need in the field for a method that will make it possible not only to unveil brain connectivity, but also to genetically modify the functional properties of the neurons connected in a circuit. We recently developed a genetic system named TRACT and showed using Drosophila that it possesses both of these features. Unfortunately, many complex brain functions cannot be examined in Drosophila, and understanding them will require studying vertebrate animals. In recent years the zebrafish has emerged as a useful animal model to study complex brain processes, because it has a relatively simple yet conserved vertebrate brain, optical transparency during embryonic and larval stages of development, amenability to large-scale behavioral assays, the emergence of complex behaviors after only 5 days of development, and a growing suite of genetic tools that allow observation and manipulation of neuronal circuits in behaving animals. However, the usefulness of zebrafish for neuroscience research is constrained by a lack of methods to identify synaptically connected neurons. Here we propose proof-of-concept experiments to establish the TRACT system for use in zebrafish to identify the wiring diagrams of brain circuits, and to genetically manipulate the functions of neurons that mediate complex behavioral states such as sleep and arousal. These capabilities will broadly increase the usefulness of zebrafish as a model system to study vertebrate neuronal circuit function, both to reveal general principles of neuronal circuits that underlie specific behaviors, and to model complex human brain disorders such as autism, Alzheimer?s disease and schizophrenia.

ABSTRACT Sleep disorders are pervasive, contribute to morbidity in several psychiatric disorders, and cause an annual economic burden of $100 billion. However, despite its importance for health, the mechanisms that regulate sleep are poorly understood. We are taking a new approach to this problem by exploiting useful features of zebrafish to answer an important and basic question: What genetic and neuronal mechanisms regulate sleep? Sleep is regulated by a homeostatic process that reflects internal cues of sleep need and a circadian process that is entrained by environmental cues and restricts sleep to the appropriate time. Sleep is also directly and rapidly regulated by a phenomenon known as masking, in which light induces wake and dark induces sleep in diurnal animals. Factors that regulate the homeostatic process have been identified, including our recent finding that the serotonergic raphe promote sleep homeostasis in zebrafish and mice. We also showed that melatonin is essential for circadian regulation of sleep in zebrafish, and identified a pathway in the brain that regulates masking. Here we build upon these discoveries to elucidate mechanisms that underlie homeostatic, circadian, and light-dependent regulation of sleep. We will investigate these mechanisms using zebrafish, a diurnal vertebrate with several advantages that complement rodent models, using a combination of genetic, optogenetic, and chemogenetic perturbations coupled with high-throughput behavioral assays and whole-brain neuronal activity monitoring with single cell resolution. In Project 1, we will identify raphe subsystems that promote sleep homeostasis, and identify genetic and neuronal circuits that act upstream and downstream of these subsystems in sleep control. In Project 2, we will identify melatonin receptors that mediate the sleep-promoting function of melatonin, and also perform a screen to identify neurons through which melatonin implements circadian regulation of sleep. Project 3 builds on our recent discovery that the hypothalamic neuropeptide prokineticin 2 suppresses both light- and dark-induced masking behavior. Similar to Project 1, we will identify genetic and neuronal circuits that act upstream and downstream of prokineticin 2 to regulate masking. In Project 4, we will validate a large number of human sleep disorder candidate genes that were identified by genome-wide association studies. We will do so by leveraging zebrafish to efficiently and inexpensively generate and test many mutant lines for sleep phenotypes. We will determine the mechanisms through which validated candidate genes regulate sleep, and integrate these genes into the pathways identified in Projects 1-3. The homeostatic (Project 1), circadian (Project 2) and light-dependent (Project 3) mechanisms that regulate sleep, as well as the sleep disorder genes identified in humans and validated in zebrafish (Project 4), are likely to be integrated at multiple levels to produce either sleep or wakefulness. This research program provides a unified platform to explore interactions between genes and neurons identified in each project. This will allow us to derive a comprehensive understanding of mechanisms that regulate sleep, and will set the stage for novel therapies for sleep disorders.

Autism spectrum disorder (ASD) is caused by both environmental and genetic factors, with the genetic contribution estimated at 60-80%. Dozens of genes that increase risk for ASD have been identified, most based on de novo mutations, but these mutations are predicted to account for only 15-20% of ASD cases. Thus, the majority of the genetic contribution to ASD is predicted to result from common and rare inherited variation, but few such genes have been identified. Recently, using whole genome sequencing, we reported genome wide evidence for >60 ASD risk genes, 26 of them novel for ASD, with signals derived from inherited and de novo protein truncating or missense mutations. The functions of most of these genes are unknown, so a crucial and necessary next step is to explore their impact on neurodevelopment and neuronal function using a model organism. The current pace of translating genetic risk factors into phenotypes, mechanisms and therapies is limited in part by inefficiencies with in vivo mammalian model systems, which makes them impractical for creating and behaviorally testing large numbers of mutant lines. Here, we leverage the zebrafish, which occupies a unique niche as a vertebrate model with features amenable to both in vivo screening and mechanistic understanding, including ex utero development, transparency, small size, rapid development, a conserved yet relatively simple vertebrate brain, behaviors relevant to ASD, and cost-effectiveness relative to mammalian models. While the zebrafish cannot recapitulate ASD and has limitations for modeling a human disorder, an emerging literature supports the notion that it is a useful model to study the functions of genes that contribute to ASD risk. Rather than assess ASD-risk genes one at a time, we will accelerate progress towards mechanistic understanding via high-throughput assays and analyses. In Specific Aim 1 we will generate null mutations in the zebrafish orthologs of 24 high confidence, novel, genome-wide significant ASD risk genes, and systematically test each mutant for neurodevelopmental, behavioral, neuronal network, and transcriptomic phenotypes. In Specific Aim 2, we will use transcriptomic analyses, at the whole brain and single cell levels, to integrate ASD risk genes into functional networks, and test for convergence across genes and species, including ASD post mortem brain. We will also test for functional associations among behavioral phenotypes that are often co-morbid in ASD, such as disrupted sleep and social behavioral deficits. In Specific Aim 3 we will perform mechanistic studies to understand how mutation of specific ASD-risk genes leads to phenotypes. This project will efficiently and cost-effectively create and characterize vertebrate animal models for a large number of novel ASD risk genes. These animal models will be a valuable resource for the community, particularly for large-scale in vivo drug screens to identify new therapies for ASD.