Thomas S. Kilduff - US grants

Cigarette smoking represents the most commonly used drug of abuse in American society. Despite extensive public education campaigns regarding the deleterious consequences of smoking, the proportion of women who smoke in North America has been estimated to be between 18-28%. In addition to active smoking, one-third to one-half of pregnant women are exposed to passive smoke. Identification of nicotine and its metabolite cotinine in hair samples of neonates indicates that human fetuses are exposed to significant levels of nicotine both through active smoking by their mothers and through maternal exposure to passive smoke. Among other abnormalities, an increased incidence of Attention Deficit Disorders and sudden infant death syndrome (SIDS) occurs in the offspring of maternal smokers. The purpose of this proposal is to evaluate the post-natal consequences of prenatal nicotine exposure in a rodent model, with particular emphasis on the development of sleep-wake and circadian systems. An underlying hypotheses of our work is that impaired or delayed maturation of the circadian and sleep/wake systems plays a role in the occurrence of SIDS. We have found that prenatal nicotine exposure in rat pups induces expression of the immediate early gene (IEG) c-fos in the fetal, but not the maternal, suprachiasmatic nucleus (SCN). Since induction of c-fos mRNA in the adult SCN by light exposure has been associated with phase-shifts of the circadian system, our results suggest that nicotine exposure may induce a desynchrony between the circadian timekeeping system of the mother and the fetus. To test this hypothesis and to further elucidate the signal transduction pathway in the SCN, we will determine the molecular basis for the differential response of the fetal vs. maternal SCN to prenatal nicotine exposure. We hypothesize that this differential response may be due to differential composition of the nicotinic cholinergic receptor in the fetal and adult SCN. To further understand the signal transduction pathway underlying nicotine-induced c- fos expression in the fetal SCN, we will determine whether prenatal nicotine exposure causes phosphorylation of the CREB and/or SRF proteins in the fetal SCN and identify other IEGs induced in the fetal SCN by prenatal nicotine exposure.

DESCRIPTION (provided by applicant): Since the initial funding of this grant proposal, the hypocretin/orexin system has been recognized to be a hypothalamic neuropeptide system with widespread excitatory activity. Defects in this system, either presynaptically or postsynaptically, result in the sleep disorder narcolepsy in both animal models and in humans. Based on these and other observations, hypocretin (Hcrt) is now viewed as a central neurotransmitter system in the maintenance of wakefulness. During the next funding period, we propose to examine specific inputs to and outputs from the Hcrt neurons at both the cellular and behavioral levels. We hypothesize that there is an interaction between the Hcrt and the corticotropin releasing factor (CRF) systems. To further understand this relationship, we will (1) determine the origin of CRF-containing afferent innervation of the Hcrt neurons using anterograde tracing from potential CRF afferent sources combined with Hcrt immunohistochemistry and retrograde tracing from the perifornical hypothalamus with CRF-immunohistochemistry; (2) identify the mechanism of action of the excitatory effect of CRF on Hcrt cells; and (3) determine whether an intact Hcrt system is necessary for the anxiogenic effects of CRF. We will identify whether CRF is anxiogenic in hypocretin-ataxin cell knockout mice and evaluate the effect of hypocretin peptides in four anxiety models in wildtype mice. These studies will determine whether the Hcrt system is "downstream" from CRF. We will also determine the response of Hcrt neurons to stimulation by other neurotransmitters that may provide modulatory input to the Hcrt system. We will examine the mechanisms underlying GABAergic inhibition of the Hcrt neurons and determine whether endogenous inhibitory tone exists in this system. We will also examine other potential sources of modulatory input to the Hcrt cells, such as that from the hypothalamic ghrelin and galanin systems. Lastly, we will determine whether subpopulations of Hcrt neurons can be differentiated based on their efferent projections to sleep-related monoaminergic and cholinergic nuclei. To address this question, we will use dual retrograde tracing from different sleep-related regions to determine the extent of co-localization of retrograde markers within Hcrt neurons and the distribution of these markers within the Hcrt neuronal population. The information obtained will elucidate the neural networks related to the Hcrt system and thereby further our understanding of the neurobiological bases of sleep, wakefulness, and the sleep disorder narcolepsy.

[unreadable] DESCRIPTION (provided by applicant): Narcolepsy, a Rapid Eye Movement (REM) sleep-related disorder, is characterized by excessive daytime sleepiness (EDS), cataplexy (a sudden loss of muscle tone triggered by emotional stimulation), and a cluster of other symptoms. Narcolepsy afflicts approximately 1 in 1000 Americans, and current treatments are symptomatic: cataplexy has classically been treated with anticholinergics, which have such undesirable side- effects that patients elect to live with cataplexy; and the debilitating sleepiness has been treated with amphetamine and other stimulant medications, which have abuse potential. More recently, modafinil has been used as a wakefulness-promoting therapeutic, and gamma-hydroxybutyrate, a controlled substance, has been approved to treat both cataplexy and EDS symptoms. Recently narcolepsy been shown to be a neurodegenerative disorder in which the hypocretin (orexin)-containing neurons of the hypothalamus specifically degenerate. The hypocretin peptides (Hcrt1 and Hcrt2) differentially bind two G protein-coupled receptors known as Hcrt receptor 1 and 2 (HcrtR1 and HcrtR2). The onset of narcolepsy symptoms is associated with a decline in Hcrt1 levels in cerebrospinal fluid, thought to be indicative of the ongoing loss of Hcrt-containing neurons in the PLH. Thus, although Hcrt replacement is a potential therapeutic regimen, no small molecule brain-penetrable HcrtR agonists currently exist. By screening a compound library synthesized at SRI International for potential HcrtR agonists, we have identified two hits, SRI-2757 and SRI-5653, that can serve as the basis for the iterative process of optimization using rational drug design and molecular modeling followed by drug synthesis and biological testing. Aim 1 will establish a structure-activity relationship for SRI-2757 and SRI-5653 and develop ligand-derived pharmacophore models and pharmacophore-based in silico screening of small-molecule libraries to identify other HcrtR agonists with novel scaffolds. Aim 2 will determine compound activity in vitro using FLIPR-based HcrtR functional assays and will evaluate the intestinal permeability and metabolic stability of selected analogs in models in vitro. Aim 3 will evaluate the efficacy of Hcrt agonists in vivo using behavioral assays in a mouse genetic model of narcolepsy. Since the Hcrt system is now recognized to be centrally involved in the maintenance of wakefulness, novel Hcrt agonists identified in this proposal may have broader indications than treating narcolepsy; for example, they may be used to prolong wakefulness in situations where sustained performance is necessary. Since Hcrt1 appears to have antinociceptive properties, Hcrt agonists may also be useful when analgesia is necessary, such as in acute injury, arthritis, and postoperative or neuropathic pain. In addition to these potential therapeutic roles, subtype-specific Hcrt agonists would be useful to help elucidate the biological functions of HcrtR1 and HcrtR2, since all reports to date have focused on the endogenous ligands, Hcrt1 and Hcrt2, and a few peptide analogs. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The hypocretin/orexin (Hcrt) system is a neuropeptide system involved in behavioral arousal, metabolism, addiction, stress-induced analgesia (SIA) and neuroendocrine function. Hcrt deficiency results in narcolepsy, a CNS disorder characterized by impaired alertness, excessive sleepiness, restricted social activities, and increased body mass index. Given the functional significance of this system, identification of the inputs that control the Hcrt system and functional Hcrt efferent pathways is of clinical relevance. We will use a combination of optogenetics, transgenic mice in which light-sensitive proteins are expressed specifically in Hcrt neurons, and conditional transgenic mouse strains to further understand the Hcrt system. Using a novel transgenic mouse that expresses the light-sensitive proton pump Archaerhodopsin-3 specifically in Hcrt neurons, we will test the hypotheses that: (1) optogenetic inactivation of the Hcrt neurons will induce changes in cortical activity, muscle tone and behavior indicative of sleep, and (2), that homeostatic sleep pressure modulates the efficacy of optogenetic inactivation of Hcrt neurons to induce sleep. Using a transgenic mouse in which the 5HT1A receptor is conditionally overexpressed in Hcrt neurons, we will test the hypothesis that the negative feedback loop between Hcrt neurons and the serotonergic dorsal raphe nucleus has functional significance for the regulation of sleep and wakefulness. Lastly, we will exploit a newly created transgenic mouse in which conditional expression of diphtheria toxin A (DTA) occurs specifically in Hcrt neurons to establish and validate a novel mouse model of human narcolepsy that more closely resembles the human disorder. The research proposed here represents an integrated systems neurobiology approach to elucidate control of a neurotransmitter system implicated in a number of behaviors and whose pathology is clearly linked to CNS dysfunction and may lead to novel pharmacotherapeutic approaches for the treatment of disorders of Hcrt insufficiency such as narcolepsy.

DESCRIPTION (provided by applicant): The trace amines (TAs), endogenous amino acid metabolites previously considered false neurotransmitters, have recently been shown to act as endogenous ligands for trace amine-associated receptor 1 (TAAR1). TAAR1 is a G protein-coupled receptor that modulates dopaminergic, serotonergic and, possibly, glutamatergic activity. In papers recently published in Molecular Psychiatry and Biological Psychiatry with scientists from F. Hoffmann-LaRoche, we describe novel, brain-penetrable TAAR1 agonists with pro-cognitive, antidepressant- and antipsychotic-like properties, suggesting TAAR1 as a novel target for the treatment of neuropathological disorders. We also show that TAAR1 partial agonism causes a dose- dependent increase in wakefulness and decreases in NREM and REM sleep, indicating that this receptor activates an endogenous wake-promoting system. In the present proposal, we will determine whether endogenous TAAR1 tone contributes to the normal distribution of sleep and wakefulness, the homeostatic response to sleep deprivation, and the response to endogenous and exogenous compounds known to promote wakefulness. First, we will determine whether TAAR1 signaling is involved in the maintenance of daily sleep- wake patterns and the homeostatic regulation of sleep in TAAR1 null mutant mice. In the context of these studies, we will determine whether the wake-promoting effects of TAAR1 agonists studied to date are absent in these mice. Next, we will determine the consequences of overexpression of TAAR1 on the normal distribution of sleep and wakefulness and the homeostatic response to sleep deprivation. Lastly, we will determine whether TAAR1 signaling is necessary for the wake-promoting effects of the stimulant caffeine and the wake- promoting therapeutic modafinil (Provigil(R)) and the endogenous wake-promoting neuropeptide, hypocretin-1 (orexin-A). The results obtained will advance our understanding of the interaction of TAAR1 with wakefulness- promoting systems in the brain, and will likely impact the development of pharmacotherapies directed toward this novel target for the treatment of sleep/wake and other neural disorders.

DESCRIPTION (provided by applicant): The goal of this proposal is to determine whether ligands for Trace Amine-associated Receptor 1 (TAAR1) are wake-promoting and pro-cognitive in non-human primates as we have found in laboratory rodents. Recent identification of selective agonists for TAAR1 that exhibit pro-cognitive, antidepressant- and antipsychotic-like properties in both rodent and non-human primates suggest TAAR1 is a novel target for the treatment of neuropsychiatric disorders. In collaboration with scientists from F. Hoffmann-La Roche, we have described brain-penetrable compounds with high potency and selectivity at mouse, rat, monkey and human TAAR1. Moreover, we showed that TAAR1 agonism causes a dose-dependent increase in wakefulness in rats and have replicated this effect in mice. Given the widespread occurrence of sleep disorders, we will further test the hypothesis that TAAR1 agonism is a novel therapeutic pathway to promote wake and enhance cognition. First, we will determine whether TAAR1 agonism promotes wakefulness in Cynomolgus macaques under conditions of both high and low sleep pressure. Although TAAR1 agonists increase vigilance in rodents, these animals are nocturnal and have polyphasic sleep/wake cycles in comparison to the consolidated periods of sleep and wakefulness characteristic of both humans and non-human primates. Implementation of EEG recording in these studies will enable us to determine the effects of TAAR1 agonism on NREM and REM sleep and to conduct quantitative EEG (qEEG) analysis to assess the effects of these compounds on EEG frequencies associated with cognition such as gamma oscillations. Next, we will test these compounds for their ability to improve working memory functions in macaques under baseline and sleep deprivation conditions. Working memory function is dependent on prior sleep history and subject to decline in aging, stress and in diseases such as schizophrenia and Alzheimer's disease. We have shown previously that TAAR1 agonists improve executive functions (e.g., response inhibition, planning) in non-human primates and in a rodent PCP-induced deficit model, however, this represents only one cognitive domain that can be affected in sleep disorders. Simultaneous assessment of behavior and qEEG in macaques performing the working memory task will enable investigation of cognitive function at both behavioral and electrophysiological levels. The results of these studies will aid in establishing TAAR1 agonism as a novel mechanism to enhance wakefulness and cognition in humans.

Melanin-concentrating hormone (MCH) and hypocretin/orexin (HCRT)-expressing neurons are intermingled populations in the tuberal hypothalamus that project widely throughout the brain to many of the same terminal fields. Whereas the HCRT system has been implicated in the control of wakefulness because the sleep disorder narcolepsy results when these cells degenerate, this system is also involved in energy metabolism. Conversely, the MCH system has primarily been associated with food intake and energy metabolism, but recent studies have established that MCH neurons also participate in the regulation of sleep and wakefulness. The hypothesis underlying this proposal is that the HCRT system is wake-stabilizing and REM-inhibiting whereas the MCH system is sleep-facilitating and REM-stabilizing. We will test this hypothesis by determining the phenotype of mice in which either the HCRT or MCH neurons have been partially ablated by removal of doxycycline in the diet of two conditional mouse models. We will then evaluate whether partial ablation of the HCRT neurons results in a phenotype of narcolepsy without cataplexy and whether cataplexy is exacerbated by simultaneously eliminating both neuronal populations. We will also assess whether direct connectivity exists between these cell groups using optogenetically-assisted neuroanatomical tracing and whole-cell patch- clamp electrophysiology in the presence and absence of selective HCRT and MCH receptor antagonists. To assess what occurs in the brain when the HCRT neurons degenerate as in human narcolepsy, we will use the conditional HCRT neuron ablation model to determine how the excitability of the MCH population is affected by chronic loss of HCRT input. We will also use conditional MCH neuron ablation to assess the converse effect of MCH loss on HCRT neuron excitability. Based on recordings from a limited number of cells in head-fixed animals, the HCRT and MCH neurons have been reported to have reciprocal activity across the sleep-wake cycle with HCRT neurons having their highest firing rates during active wakefulness and MCH neurons being primarily active during REM sleep. To determine the accuracy of this conclusion, we will use genetically- encoded Ca2+ indicators and microendoscopic imaging to measure the activity of hundreds of HCRT and MCH neurons across the sleep/wake cycle in unrestrained, freely-moving animals. To evaluate whether the HCRT and MCH neurons are functionally interconnected, we will pharmacogenetically activate one population while imaging Ca2+ fluorescence in the other population in the presence of selective HCRT and MCH receptor antagonists. Lastly, since these two populations project to many of the same brain regions, we will assess their relative input to brain areas known to be involved in arousal state control, specifically, the locus coeruleus, tuberomammillary nucleus, medial septum, and the amygdala. Together, these experiments should provide a more complete picture of the anatomical and functional connectivity of these two populations and the consequences of selective loss of one population or the other.

PROJECT SUMMARY The hypocretin/orexin (Hcrt) system is a hypothalamic neuropeptide system that is involved in behavioral arousal, metabolism, addiction, stress and neuroendocrine function. Defects in this system, either presynaptically or postsynaptically, result in narcolepsy in both humans and animals. A convergence of results led to the recognition that human narcolepsy is a neurodegenerative disorder in which the Hcrt neurons, located exclusively in the perifornical and lateral hypothalamus, specifically degenerate. Thus, narcolepsy is due to Hcrt insufficiency. The Hcrts are a pair of peptides that differentially bind the G protein-coupled receptors Hcrt receptor 1 (HcrtR1 or OX1R) and HcrtR2 or OX2R. Based in part upon the excessive daytime sleepiness (EDS) that characterizes narcolepsy, it was recognized that the Hcrt system is centrally involved in the maintenance of wakefulness, which led to the development of HcrtR antagonists for the treatment of insomnia and FDA approval of suvorexant (Belsomra?) in 2015. In contrast, current narcolepsy treatments are purely symptomatic: cataplexy is treated with antidepressants or anti-cholinergics which have undesirable side- effects, and the debilitating EDS is often treated with amphetamine or other stimulants which have abuse potential. The goal of our research is to develop HcrtR/OXR-directed narcolepsy therapeutics that are both wake-promoting and cataplexy-inhibiting. Based on the screening of UT Southwestern's ~220,000-compound library, we have developed new HcrtR/OXR agonists with novel scaffolds and carried out structure-activity relationship studies to improve their potencies. Having optimized these initial HcrtR/OXR agonist hits using an iterative process of analog synthesis, biological testing, and molecular modeling using HcrtR/OXR crystal structures, we will now determine the pharmacokinetic (PK) properties of drug-like small molecule HcrtR/OXR agonists to select the best candidates for in vivo efficacy experiments. Thus, we will conduct PK studies on each molecule for metabolic stability, intestinal permeability, plasma concentration over time, and blood-brain- barrier (BBB) permeability using a combination of in vitro and in vivo assays. We will then test the most promising compounds in series of in vivo screens, culminating in evaluation in orexin/tTA; Tet-O DTA mice, a state-of-the-art narcolepsy model in which ablation of Hcrt neurons is controlled through the tetracycline transactivator (Tet-off) system. Using this model, the effects of HcrtR/OXR agonists on arousal state will be evaluated before and after induction of Hcrt cell loss to determine whether HcrtR/OXR agonism normalizes the narcoleptic phenotype; dose-response tests will also be conducted in the DTA model. Given that the Hcrt system is centrally involved in the maintenance of wakefulness, Hcrt agonists may have a broader indication than narcolepsy, e.g., to prolong wakefulness in civilian, industrial or military situations where sustained alertness is required. In addition to these and other potential therapeutic indications, subtype-specific Hcrt agonists would be useful to help elucidate the biological functions of HcrtR1/OX1R and HcrtR2/OX2R.

PROJECT SUMMARY Melanin-concentrating hormone (MCH) and hypocretin/orexin (HCRT)-expressing neurons are intermingled populations in the tuberal hypothalamus that project widely throughout the brain to many of the same terminal fields. Whereas the HCRT system has been implicated in the control of wakefulness because the sleep disorder narcolepsy results when these cells degenerate, the HCRT system is also involved in energy metabolism. Conversely, the MCH system has primarily been associated with food intake and energy metabolism, but recent studies have established that MCH neurons also participate in the regulation of sleep and wakefulness. The overarching hypothesis of the Parent R01 grant (R01 NS098813 ?The Tuberal Hypothalamus and Arousal State Control?) is that the HCRT system is wake-stabilizing and REM-inhibiting whereas the MCH system is sleep-facilitating and REM-stabilizing. We will test this hypothesis by determining the phenotype of mice in which either the HCRT neurons, MCH neurons or both cell types have been ablated by removal of doxycycline from the diet of two conditional mouse models, orexin-tTA;TetO DTA mice and MCH-tTA;TetO DTA mice. Professor Yamanaka's lab at Nagoya University has recently created two novel mouse strains that would be quite valuable for us in executing the Aims 1b, 2 and 3 proposed in the Parent R01 grant to test the overarching hypothesis. Specifically, his lab has crossed the orexin-tTA;TetO DTA mice with MCH-tTA mice to create an orexin-tTA;MCH-tTA;TetO DTA mouse strain in which both the orexin and MCH neurons can be conditionally ablated by removal of doxycycline. This strain of double-ablated neuron mice is critical for Aim 1 of ?The Tuberal Hypothalamus and Arousal State Control? to determine whether cataplexy is exacerbated by simultaneously eliminating both neuronal populations. The second novel mouse strain that Professor Yamanaka's lab has generated is a knock-in (KI) mouse in which Flp recombinase is expressed exclusively in the orexin neurons (orexin-Flp mice), which will be extremely valuable for us to address Aims 2 and 3 of NIH R01 NS098813 ?The Tuberal Hypothalamus and Arousal State Control?. An Administrative Supplement from the US-Japan Brain Research Cooperative Program would also enable these two laboratories to establish collaboration on a new topic: investigation of the role of MCH neurons in memory consolidation during sleep. Together, these experiments should provide a more complete picture of the interaction of the HCRT and MCH neuronal populations, the consequences of simultaneous loss of these cells, and the hypothetical role of MCH neurons in memory.