Philippe Mourrain, Ph.D. - US grants

Melanin-concentrating hormone: ancestral role in feeding &sleep regulation In mammals, melanin-concentrating hormone (hMCH) is a key regulator of feeding behavior, energy homeostasis, and sleep. MCH was first identified in salmon in 1983 as a peptide (sMCH) that induced skin lightening. Despite numerous studies in different fish species, however, no clear effect on feeding was shown for sMCH. Last year, we found, in zebrafish and four other teleost fishes, two MCH genes: mch1 and mch2. Whereas mch1 perfectly resembles salmon sMCH, the mch2 gene and MCH2 peptide share genomic structure, synteny, and high homology with mammalian hMCH. Zebrafish MCH2, like mammalian hMCH, is expressed in a distinct population of hypothalamic neurons and is up-regulated upon fasting, suggesting a conservation of MCH2/hMCH regulation and function across vertebrates. However, while mammalian hypothalamus harbors thousands of MCH cells, zebrafish larval and adult brains contain more compact networks of 50 and 150 MCH neurons respectively. As MCH is not found in non-vertebrate models like Drosophila and C. elegans, the discovery of mch2 ofers us a unique opportunity to explore MCH function in a simple amenable genetic model, the zebrafish. We propose in a first specific aim to characterize the MCH2 neurocircuit and to relate its activity to behavioral states. To do so, we will (i) analyze MCH2 neurons identity and fast neurotransmitter phenotype, (ii) study their arborization and connections, and (iii) precisely monitor their pattern of activity with a calcium-imaging assay in different behavioral conditions. As feeding and sleep behaviors are exclusive in their timing, it is critical to follow the firing patterns of the totality of the MCH neurons to distinguish potential feeding- related and sleep-on subpopulations. In the second specific aim, we will precisely investigate the behavioral influence(s) of MCH2. To do so we will use both a classic genetic approach and state-of-the- art optogenetic manipulation of the mch2 circuit. We will first (i) study how the MCH2 peptide and circuit regulate food intake, growth, and body weight. Further, (ii) using a novel multi-behavioral tracking system we will analyze a large spectrum of behaviors associated with energy unbalance, food search and consumption, such as exploration, anxiety, aggressiveness, bulimic like behaviors vs. slow eating, and the hedonistic influence of food. Finally, (iii) we will investigate the ancestral role for MCH in sleep- wake regulation by analyzing the sleep architecture during normal sleep, induced sleep, and after sleep deprivation. In conclusion, zebrafish offers a unique situation as a transparent vertebrate to perform non-invasive observation and manipulation of small but complete neuronal networks. It will bring major insight to our understanding of how a same hypothalamic circuit times and regulates behaviors so distinct in their timing and their functions.

DESCRIPTION (provided by applicant): Sleep is a fundamental biological process conserved across the animal kingdom. Zebrafish, a small diurnal teleost extensively used in developmental biology, will be used as a model to study sleep and sleep regulatory networks. Previous studies have shown that a genuine sleep-like state exists in this species, as defined using behavioral criteria (circadian rhythm, reversible periods of immobility, place preference, characteristic posture, increased arousal threshold, sleep rebound). Moreover, sleep-wake molecular actors identified in mammals are also conserved in this species as well as hypnotic drug targets. Finally, the hypocretin (hcrt, aka orexin) system, a system involved in the pathophysiology of the sleep disorder narcolepsy, also exists in zebrafish. HCRTs are neuropeptides involved in the regulation of sleep and energy balance in mammals. We found that there are less than 50 hypocretin neurons in an adult zebrafish hypothalamus and cloned a very compact zebrafish hcrt promoter (1kb) capable of accurately mimicking the native hypocretin pattern. Moreover, we also identify a null mutation (hcrtr168) in the sole hypocretin receptor (hcrtr) present in zebrafish. Fish lacking this receptor have fragmented sleep and a 30% sleep reduction in the dark. In this proposal, we propose to identify, with a yeast one-hybrid assay, transcription factor(s) able to bind a core 13 base pair promoter element essential and sufficient for hypocretin expression (first specific aim). Further, we plan to study the zebrafish hcrt neurocircuitry to understand its sleep-promoting function (second specific aim). To do so, we will, (i) study its connections with hcrt:GFP and hcrtr:mCherry transgenic lines, (ii) study when it is active with a calcium imaging assay using hcrt:GCaMP2 and hcrtr:GCaMP2 transgenic lines, and (iii) evaluate whether hypocretin signaling is excitatory or inhibitory by analyzing neurotransmitter phenotype co-expression and the activity of the hcrtr positive neurons (hcrtr:GCaMP2) when hcrt neurons are silenced (hcrt:Kir2.1) or the hcrtr is missing (hcrtr168). Finally in the third specific aim, we will identify novel sleep- and wake-active nuclei in this species using immediate early gene (c- fos) expression analysis of adult zebrafish brains during the night and the day, after sleep deprivation and after hypnotic drug treatments. PUBLIC HEALTH RELEVANCE 15% of the population suffers of sleep disorders. However, sleep is still a poorly understood phenomenon. Our laboratory uses a simple model animal, the zebrafish, to understand the development, organization and function of a neuronal hypocretin system, responsible when disrupted of the sleep disorder Narcolepsy. As a developmental model, zebrafish will help us to understand how hypocretin expression can be lost in humans and, and as a neurobiology/sleep model, it will help us to decipher the underlying molecular and cellular mechanisms of sleep, and to generate the basic knowledge indispensable for future efficient therapies.

DESCRIPTION (provided by applicant): In mammals, melanin-concentrating hormone (hMCH) is a key regulator of feeding behavior, energy homeostasis, and sleep. MCH was first identified in salmon in 1983 as a peptide (sMCH) that induced skin lightening. Despite numerous studies in different fish species, however, no clear effect on feeding was shown for sMCH. Last year, we found, in zebrafish and four other teleost fishes, two MCH genes: mch1 and mch2. Whereas mch1 perfectly resembles salmon sMCH, the mch2 gene and MCH2 peptide share genomic structure, synteny, and high homology with mammalian hMCH. Zebrafish MCH2, like mammalian hMCH, is expressed in a distinct population of hypothalamic neurons and is up-regulated upon fasting, suggesting a conservation of MCH2/hMCH regulation and function across vertebrates. However, while mammalian hypothalamus harbors thousands of MCH cells, zebrafish larval and adult brains contain more compact networks of 50 and 150 MCH neurons respectively. As MCH is not found in non-vertebrate models like Drosophila and C. elegans, the discovery of mch2 offers us a unique opportunity to explore MCH function in a simple amenable genetic model, the zebrafish. We propose in a first specific aim to characterize the MCH2 neurocircuit and to relate its activity to behavioral states. To do so, we will (i) analyze MCH2 neurons identity and fast neurotransmitter phenotype, (ii) study their arborization and connections, and (iii) precisely monitor their pattern of activity with a calcium-imaging assay in different behavioral conditions. As feeding and sleep behaviors are exclusive in their timing, it is critical to follow the firing patterns of the totality of the MCH neurons to distinguish potential feeding- related and sleep-on subpopulations. In the second specific aim, we will precisely investigate the behavioral influence(s) of MCH2. To do so we will use both a classic genetic approach and state-of-the-art optogenetic manipulation of the mch2 circuit. We will first (i) study how the MCH2 peptide and circuit regulate food intake, growth, and body weight. Further, (ii) using a novel multi-behavioral tracking system we will analyze a large spectrum of behaviors associated with energy unbalance, food search and consumption, such as exploration, anxiety, aggressiveness, bulimic like behaviors versus slow eating, and the hedonistic influence of food. Finally, (iii) we will investigate the ancestral role for MCH in sleep- wake regulation by analyzing the sleep architecture during normal sleep, induced sleep, and after sleep deprivation. In conclusion, zebrafish offers a unique situation as a transparent vertebrate to perform non-invasive observation and manipulation of small but complete neuronal networks. It will bring major insight to our understanding of how a same hypothalamic circuit times and regulates behaviors so distinct in their timing and their functions. PUBLIC HEALTH RELEVANCE: In the zebrafish species, we have recently identified the equivalent of a major mammalian feeding and sleep hypothalamic actor called Melanin-Concentrating Hormone (MCH). The zebrafish will help us understand how MCH can regulate food consumption, energy balance, and sleep, and thus how MCH could be manipulated to prevent feeding or sleep disorders in the general public.

Abstract We still wonder why we sleep. We know at least that sleep helps our memory. Almost every stages and features of sleep are involved memory consolidation, including non-rapid eye movement slow wave sleep (NREM SWS), NREM spindles, REM theta rhythm and sleep architecture continuity. Disruption of these stages and features are found in all neurological disorders afflicting memory (Angelman, autism spectrum, alcoholism, Alzheimer's, fragile X, Huntington's, Parkinson's, Rett etc?). The mechanisms underpinning these memory deficits are poorly understood and the role of sleep at the synapse is still highly debated. Synpases are the central physiological structures underpinning memory and cognition, but how each sleep stages and features remodels synapses remains unclear. NREM SWS and total sleep have been implicated in general synaptic downscaling, but NREM and spindles have also been involved in synaptic strengthening; similarly REM has been associated to both synapse pruning and maintenance. One major obstacle to such study has been that sleep stages and features are all interconnected and integrated. The disruption of one often impacts the others making the association between a stage/feature and a specific synaptic function challenging. Using precise optogenetic control of neuronal circuits, we have overcome this obstacle. Sleep continuity and memory consolidation can be disrupted without changing overall sleep architecture and quantity by introducing micro-arousals (<2sec) every 60 sec using hypocretin neuron stimulation (Aim 1). NREM sleep spindles and memory consolidation can be elicited by stimulating reticular thalamus neurons without disturbing sleep (Aim 2). Theta rhythms and memory consolidation can be disrupted by silencing medial septum GABA neurons during REM bouts only without affecting sleep architecture integrity (Aim 3). We will manipulate these three sleep features after a cortical motor learning task which rapidly induces synapse formation in the motor cortex. Remodeling of these newly formed synapses and their neighbors will be followed using state-of-the-art in vivo (two-photon) and ex vivo (array tomography) synapse microscopy. While the former longitudinal analysis will uncover the spine dynamics leading to memory encoding consolidation, the latter global synapse analysis will reveal how synapse classes (inhibitory, excitatory), synapse populations (depending on layers) and their subsynaptic molecular components are remodeled by sleep continuity (Aim 1), spindles (Aim 2) and REM specifically (Aim 3). The specific use of optogenetics to manipulate different sleep stages as synaptic dynamics are studied is unprecedented and will shed important light on how sleep continuity, NREM spindles, and REM can each influence cortical synaptic plasticity underpinning memory consolidation after motor learning. These discoveries are crucial for future strategies to recover and treat memory and cognitive deficits in neurodevelopmental and neurodegenerative disorders.

Abstract We propose to develop and apply fluorescence-based polysomnography (fPSG) in zebrafish, a novel, non- invasive method allowing neurogenetic and pharmacological interrogations of nervous system function through whole brain and whole body imaging. fPSG combines custom light sheet microscopy with a new zebrafish line ?zPSG? carrying four transgenes expressing GCaMP7a [Tg(5xUAS:GCaMP7a)] in the brain [Tg(?-tubulin:nls-Kal4FF)] and trunk muscles [Et(gSAIzGFFD109A)], and GFP in the heart [Tg(cmlc2:GFP)] in order to capture brain wide Ca2+ activity (fEEG, fluorescent electroencephalogram), muscle Ca2+ activity (fEMG, fluorescent electromyogram), heart rate (fECG, fluorescent electrocardiogram) as well as eye movement (fEOG, fluorescent electrooculogram). Polysomnography (PSG) is a classic method used to characterize sleep and diagnose sleep disorders and sleep abnormalities in neurological and psychiatric disorders. Slow wave sleep (SWS, non-REM) and rapid eye movement sleep (REM, a.k.a. paradoxical sleep, PS) are defined by specific electrophysiological PSG signatures based on recordings from the surface of the neocortex (EEG), and voluntary or autonomous muscles (EMG+ECG+EOG). SWS-REM/PS have only been reported so far in the more evolutionary-recent amniotic vertebrates: mammals, birds and reptiles. It is unclear whether such neuronal and muscular dynamics are found in non-amniotic vertebrates such as fishes and amphibians. In a first study we have found slow synchronous neural activity and traveling waves of neural activity in the sleeping fish brain. We have coined these novel signatures: Slow Bursting Sleep (SBS) and Propagating Wave Sleep (PWS) which share remarkable commonalities with SWS and PS/REM states, respectively. We propose to develop and apply fPSG to fully characterize SBS (Aim 1) and PWS (Aim 2) at the whole brain, body scale levels. After this full characterization, we will next investigate the molecular and circuit underpinning of these dynamics by interrogating different neurogenetic contexts of melanin-concentrating hormone (MCH) signaling, a conserved neuropeptidergic system which is involved in mammalian sleep but whose role in fish sleep has been debated for over 30 years (Aim 3). Overall, this proposal will (i) develop a new PSG methodology with whole brain-single cell resolution imaging and body scale comprehension that could also be used with other fish models [e.g. cavefish, danionella, medaka], (ii) establish the first neural definition of sleep in fish, (iii) uncover the role of MCH in fish sleep, and finally (iv) shed light on whether common neural signatures of sleep emerged in the non-amniotic vertebrate brain over 450 million years ago. Importantly, fPSG tools and methodology can be extended to any neuroscience question in the awake or asleep animal requiring whole brain imaging with cardiovascular, ocular and voluntary muscles readouts (e.g. studies of the autonomic and non-autonomic systems).

Project Summary The improvement in living standards and the advancement in modern medicine have greatly extended human life expectancy. However, aging-related functional decline and diseases, in particular cognitive impairment and neurodegeneration, also become more prevalent. Studies of heterochronic blood exchange reveal that the aged systemic milieu inhibits neurogenesis and impairs cognitive functions in young animals, suggesting the existence of age-elevated systemic factors detrimental to brain health. In particular, inflammation may become excessive and chronic with aging (?inflammaging?) and impair normal brain functions. Thus proteins involved in inflammatory responses, such as cytokines, are candidates of such systemic factors implicated in brain aging. Building upon published literature and our recent finding, we hypothesize that aging-associated alterations in systemic inflammatory factors activate microglia (resident immune cells in the central nervous system) and lead to microglia-mediated synapse loss; restoring the expression pattern of such factors to the healthy young state rescues synaptic defects and improves cognitive functions. In Aim 1, we will use bio-orthogonal non- canonical amino acid tagging (BONCAT) to determine how treatment with a cocktail of Alk5 inhibitor (Alk5i) and oxytocin (OT, a neurotrophic, anti-inflammatory peptide) or heterochronic blood exchange affects the expression profile and distribution of inflammaging-related systemic factors in the brain and peripheral tissues. Aim 2 examines how Alk5i+OT treatment and heterochronic blood exchange affect neuro-immune interaction in the brain, taking advantage of in vivo two-photon imaging to study microglia-synaptic interactions and their effects on synaptic integrity and dynamics in the cortex. Using Array Tomography, a high-throughput, super- resolution proteomic imaging technique, Aim 3 conducts molecular dissection and reconstruction of large populations of individual synapses and determines the effect of Alk5i+OT treatment and heterochronic blood exchange on synaptic molecular signatures and inflammatory cytokine distribution in the brain. Together, these studies will provide a comprehensive characterization of age-specific effects of blood on the brain proteome and synaptic circuits, and outline candidate mechanism(s) responsible for brain aging.

Project Summary Conserved Non-protein coding Elements (CNEs) are <1kb DNA elements deeply conserved across vertebrate genomes from zebrafish to human. While their role is not fully understood, they are prime candidates for cis- regulatory function and can act as enhancers. As some have been implicated in human biology and diseases, we developed a method to identify CNEs harboring risk SNPs identified in GWAS. Our method focused on CNE/SNPs regions deeply conserved across vertebrate genomes that also preserve gene synteny in their neighborhood to pinpoint potential cis regulated genes. Based on GWAS replications, we selected 20 CNE/SNPs pairs and their syntenic genes potentially contributing to 5 human traits (sleep/circadian activity, skin pigmentation, cardiovascular system, eye biology, body size and morphology) that can be modeled in zebrafish. Independent and in depth in vivo characterization of two CNEs (1 and 19) showed that (i) human CNE specific transcriptional enhancer activity can be revealed in live zebrafish, (ii) the risk SNP abolishes this activity, (iii) the genuine cis-regulated gene associated to the human trait can be discovered, and (iv) the underpinning human biology can be identified and studied by modeling the genetic defect in zebrafish. Based on these successful validations and the exciting promise of shedding light on the molecular and cellular biology underpinning human biological traits, we propose to test the central hypothesis that deeply conserved non-coding SNPs are regulatory genetic variants responsible for differences in gene expression and function that affect human health. This hypothesis will be tested via the following specific aims. Aim 1 will determine the transcriptional activity of the remaining 18 conserved human CNEs and associated risk SNPs in vivo, and establish the mRNA patterns of the 34 syntenic neighbor genes. Among the latters, Aim 2 will identify the actual cis-regulated genes via systematic CRISPR/Cas9 editing of CNEs and mRNA (dys)regulation analysis. Finally, Aim 3 will identify the genetic and biological consequences of disrupting CNEs (deletion, introduction of risk SNP) and their cis- regulated genes (indels). Aim 1 will use transgenesis in zebrafish to demonstrate that human CNEs are enhancers whose functions are disrupted by the risk SNPs. Aim 2 will use CRISPR/Cas9-based genome editing in zebrafish to delete all 18 CNEs (DCNE) or introduce risk SNPs in the zebrafish genome (CNE*) to identify the syntenic neighbor genes that are cis-(dys)regulated. Aim 3 will compare the consequences of enhancer mutants (DCNE, CNE*) with cis-regulated gene mutants to uncover the mechanisms underpinning the human biology and traits. The approach of using high-throughput CRISPR/Cas9-mediated genome editing in zebrafish to uncover the functional relevance of human CNE/SNPs is innovative. The proposed research is expected to be significant because it will establish the functional impact of non-coding genetic variants in human traits/diseases and will shed light on the associated human biology with in vivo genetic modeling in zebrafish.