Steven D. Munger, P.D. - US grants

The proposed research is aimed at characterizing a specialized subset of neurons in the olfactory epithelium. These D neurons appear to be unique in expressing a novel signal transduction pathway (guanylyl cyclase, GC- D) among olfactory receptor cells. These neurons may also be involved in mediating suckling behavior in neonatal rodents. The applicant proposes to: 1) isolate D neurons and characterize the signal transduction pathway and receptor molecules expressed in these cells; 2) show that GC-D is necessary for the normal expression of tyrosine hydroxylase (TH) in the olfactory bulbs, as an indicator of afferent activity; 3) examine the projection patterns of D neurons; and 4) show that GC-D neurons are necessary for nipple attachment and suckling in neonatal mice. These aims are related in an attempt to associate a specific subset of olfactory receptor cells, with unique signal transduction, to a particular set of neonatal behaviors. GC-D knockout/knockin mice will be generated to carry out the proposed experiments to identify D neurons, which make up less than 1% of olfactory receptor cells. A variety of molecular biological techniques will be used to screen isolated D neurons for the receptors and signal transduction components and use histochemical techniques for identifying projection patterns of D neurons and TH in the olfactory bulbs. In addition, GC-D transgenic mice will be used to examine the ability of neonates to attach to nipples and suckle.

DESCRIPTION (provided by applicant): Mammals use several chemosensory systems to detect and encode their chemical environment. How these systems discriminate relevant chemical cues is a major unresolved question. We hypothesize that differences in the stimulus selectivity of different populations of chemosensory cells largely reflects differences in the ligand selectivity and sensitivity of the chemosensory receptors (CRs) expressed therein. Difficulties in obtaining large amounts of receptor protein suitable for biochemical or structural analysis, as well as the small number of CRs for which ligands are known, has hampered efforts to characterize the basis of ligand specificity. One group of CRs, the T1R taste receptors, offers unique advantages that will permit the first systematic analysis of how CR structure/function relationships impact the ability of a chemosensory cell population to detect and discriminate physiologically relevant ligands. We will take advantage of the demonstrated sensitivity of T 1Rs for sweet-tasting ligands, and an extracellular N-terminal ligand-binding domain amenable to biochemical purification and structural characterization, to establish the role of different T1Rs in the detection of sweet tasting stimuli. Aim 1: The structure of the T1R ligand-binding pockets, in the presence and absence of ligands, will be solved by a combination of circular dichroism spectrophotometry and X-ray crystallography of T1R N-terminal domains. Aim 2: To determine the specific contributions of ligand binding to taste function, targeted mutations will be introduced in the ligand-binding pocket of T1R N-terminal domains both in vitro and by gene targeting in mice. Changes in ligand binding kinetics will be measured using isothermal titration calorimetry, while the effects of T1R deletion or mutation on taste function will be assayed by brief-access behavioral tasks where the sensitivity of targeted mice to sweet stimuli will be determined. Together, these studies will provide the first in-depth structural and quantitative analyses of the interactions between chemosensory receptors and their ligands, and will offer important new insights into how individual taste receptors contribute to the detection and discrimination of food cues critical for health and survival.

DESCRIPTION (provided by applicant): The sense of smell is the principal window on the rich and complex world of volatile chemicals. The main and accessory olfactory systems of mammals serve to detect and analyze odors and pheromones. However, it is becoming clear that the division of nasal chemosensory neurons into only these two groups is simplistic and does not reflect the functional diversity of receptor cell types in the nose. A novel group of chemosensory neurons, which target a unique population of glomeruli, the "necklace" glomeruli, in the posterior olfactory bulb, are found in several regions of the main olfactory epithelium. These neurons also exhibit a gene expression profile distinct from that of other chemosensory neurons. However, it is unclear if these receptor neurons and glomeruli function as a distinct chemosensory system. We have created a gene-targeted mouse that functionally disrupts the gene encoding the guanylyl cyclase, GC-D, a protein critical for the function of these cells. After targeting, this gene locus also encodes a histochemical reporter, tau-beta-galactosidase that permits the visualization of the neurons that express GC-D. We propose to use this mouse model to perform the first systematic analysis of the organization and functional characteristics of this poorly understood population of chemosensory neurons, as well as to characterize the functional role of these cells in chemosensory behavior. We will provide a systematic anatomical description of these neurons and the necklace glomeruli and determine the developmental and anatomical expression patterns of GC-D in the olfactory system. We will also determine the chemosensory role of these neurons through behavioral and biochemical analyses of adult and neonatal animals. Finally, we will characterize the biochemical cascade involved in the transduction of specific chemosensory stimuli, including both odors and pheromones, in these cells. These results obtained in these studies will provide important insights into the mechanism by which these neurons and glomeruli detect and analyze sensory stimuli, the role of cGMP signaling in these processes, and will define the sensory role of this unique neuronal population.

DESCRIPTION (provided by applicant): A key function of the gustatory system is to detect nutrients, toxins and indicators of spoilage, thus providing critical information to the animal about the quality and nutritional value of food before it is ingested. The ability to detect and discriminate taste stimuli is essential for health and survival, and can drive ingestive behaviors. Therefore, physiological mechanisms that modulate taste function in the context of nutritional needs and metabolic status could optimize ingestive decisions and directly impact human health. Although the gustatory system critically influences food preference, food intake and metabolic homeostasis, the physiological mechanisms that link taste function and metabolism are poorly understood. Recent findings from our laboratory and others suggest that the gustatory and gastrointestinal systems utilize a common molecular toolkit of receptors, signaling molecules and hormones to detect nutrients and other chemicals. This is consistent with a role for taste function in the maintenance of metabolic homeostasis and suggests that sensory function may be modulated in the context of metabolic status. A greater understanding of the interactions between metabolic hormones and the gustatory sensory apparatus would have important consequences for understanding the etiology of metabolic disease. This project will investigate the interactions between taste and hormonal systems in three Aims. In Aim 1, we will examine the contribution of taste receptors and taste receptor variants to the maintenance of glucose homeostasis. In Aim 2, we will characterize the role of two hormones expressed in taste cells and in the digestive system, glucagon-like peptide-1 (GLP-1) and glucagon, in the modulation of taste sensitivity. In Aim 3, we will examine the ability of GLP-1, glucagon and another hormone, leptin, to influence each other's effects on taste function. Together, these studies will offer important new insights into the interplay between taste, nutrition and metabolism and could have broad implications for metabolic diseases such as obesity, Type 2 diabetes mellitus, and the metabolic syndrome. PUBLIC HEALTH RELEVANCE Although the gustatory system critically influences food preference, food intake and metabolic homeostasis, the physiological mechanisms that link taste function and metabolism are poorly understood. Recent findings from our laboratory and others suggest that taste is important for the maintenance of metabolic homeostasis and that taste function may be modulated in the context of metabolic status. The proposed studies will investigate the interactions between taste and hormonal systems, providing important new insights into the interplay between taste, nutrition and metabolism that could have broad implications for metabolic diseases such as obesity, Type 2 diabetes mellitus, and the metabolic syndrome.

DESCRIPTION (provided by applicant): Social interactions in humans and other mammals are significantly impacted by olfactory signals. For example, semiochemicals (chemosensory stimuli that communicate information between organisms;e.g., pheromones or social cues) can promote mating or aggression behaviors or can contribute to transmission of food preferences between individual mice. However, the molecular, cellular and neural mechanisms underlying olfactory-mediated social interactions remain poorly understood. This proposal will examine the role of a distinct olfactory subsystem within the main olfactory system, the GC-D+ neuron/necklace glomeruli subsystem, in the detection of semiochemicals and the mediation of social interactions related to food preference. GC-D+ neurons differ from canonical olfactory sensory neurons in the transduction-related proteins they express (e.g., the receptor guanylyl cyclase GC-D) and in their olfactory forebrain targets (the necklace glomeruli of the posterior main olfactory bulb). GC-D+ neurons appear to function as multimodal chemosensors, exhibiting responses to a small group of chemostimuli including urine, the natriuretic peptide hormones uroguanylin and guanylin, and to CO2. Anatomical studies suggest that necklace glomeruli also receive diverse chemosensory inputs through heterogeneous afferent innervation and extensive intrabulbar connections with other olfactory glomeruli. Thus, the GC-D/necklace subsystem may be ideally suited to integrate semiochemical and general odor information and may act as coincidence detectors for multiple chemosensory stimuli. We propose a multidisciplinary, collaborative study to investigate the role of the GC- D/necklace subsystem in the detection of semiochemicals as they relate to social interactions. Our proposed study will (1) use molecular biological, electrophysiological and Ca2+-imaging approaches to characterize the responses of GC-D+ neurons to several semiochemicals;(2) use neuroanatomical tracing, immunohistochemistry and electron microscopy to characterize those neurons that provide sensory input to the necklace glomeruli, as well as the central targets of necklace-associated projection neurons;and (3) examine contributions of GC-D+ neurons to important social behaviors, the social transfer of food preference (STFP) and food source preference. Deficits in normal social interactions are a hallmark of autism and many other neurological disorders. Because of this, assays of social interactions such as STFP have been utilized in the study of mouse models of autism. Thus, results obtained here will not only elucidate key mechanisms underlying olfactory-mediated social communication, but should provide important insights into those diseases, such as autism, that show deficits in normal social interactions. PUBLIC HEALTH RELEVANCE: Deficits in normal social interactions are a hallmark of autism and many other neurological disorders. Social interactions in humans and other mammals are significantly impacted by olfactory signals. However, the molecular, cellular and neural mechanisms underlying olfactory- mediated social interactions remain poorly understood. Results obtained here will not only elucidate the mechanisms underlying olfactory-mediated social communication, but should provide important insights into those diseases, such as autism, that show deficits in normal social interactions.

DESCRIPTION (provided by applicant): Many of the molecules that are critical for the detection and transduction of odors by olfactory sensory neurons (OSNs) have been identified, and their basic roles in these processes defined. However, significant gaps in our understanding of the olfactory transduction process remain. For example, signal transduction cascades found in many neurons function within signaling complexes composed of receptors, effector enzymes, channels, scaffolding elements and other molecules. These signalplexes may enhance both the efficiency and specificity of signaling by increasing the local concentration of signaling elements (e.g., receptors, enzymes and soluble messengers such as cAMP or Ca2+), restricting proteins to functionally important cellular domains (e.g., dendritic spines or dendritic cilia), orby regulating access of modulatory proteins (e.g., receptor kinases, ¿-arrestin). However, little is known about how olfactory transduction proteins interact to impact olfactory function. Our proposed studies will address this important yet understudied area of olfactory biology by using a cutting-edge, multidisciplinary approach to define protein-protein interactions for key components of the olfactory transduction cascade. Our studies will focus on identifying proteins that directly interact with two key olfactory transduction molecules: olfactory marker protein (OMP) and canonical odorant receptors (OR). While both OMP and the ORs have been shown to interact with other OSN proteins (Bex proteins in the case of OMP and receptor trafficking protein (RTP) family members in the case of ORs), functional studies suggests that other partners exist for both proteins. For example, OMP influences cAMP kinetics and Ca2+ dynamics in OSNs, while native ORs are hypothesized to require additional OSN-specific co-receptors or chaperones to efficiently target the plasma membrane. Recent advances in proteomics now offer a unique opportunity to both validate previously implicated OMP and OR interactors as well as to identify novel proteins that associate with these key transduction molecules. In this proposal, the P.I.s will take advantage of our complementary expertise in olfactory transduction and state-of-the-art proteomics approaches to complete two parallel Specific Aims focused on identifying protein interactors for two baits: OMP (Aim 1) and the heptanal-responsive OR I7 (Aim 2). We will use stable isotope labeling of mice expressing different levels of the either bait, followed by immunoprecipitation of interacting complexes from native olfactory epithelium and subsequent liquid chromatography-tandem mass spectroscopy to quantitatively isolate specific interactors with high sensitivity. These studies will result in significant advances in our understanding of odor detection and transduction, and will establish an important new approach for characterizing the interactions of rare proteins in nearly any biological system.

DESCRIPTION (provided by applicant): Two paradigm-shifting discoveries in taste research in recent years have realigned our thinking as to how taste perception is linked to mechanisms of appetite and satiety. The first was that many cells in the gut express the same molecular machinery required for nutrient detection as that found in taste cells. We now know these receptors in the gut detect ingested nutrients and mediate the secretion of gastric hormones. More recently, it was learned that these 'gastric' hormones together with their cognate receptors are also expressed in taste cells in the peripheral gustatory system. This latest discovery has raised a fundamental challenge to the field to understand how these peripheral 'gastric' hormones affect taste function and ingestive behavior. Given the worldwide rising incidence of diabetes, obesity and related metabolic disorders, we are proposing research that addresses our dearth of knowledge regarding the hormonal modulation of chemosensory perception and how disruption of hormonal signaling in the taste system can impact upon food intake and energy homeostasis. We have recently reported that the gut hormone, glucagon-like peptide 1 (GLP-1), can modulate sweet taste sensitivity. We have also reported that another GI peptide, glucagon, which plays a major role in the regulation of glucose homeostasis, can also act to modulate taste sensitivity to sweeteners. These results, when placed in the context of our preliminary findings on the hormone peptide YY (PYY) suggest that the gustatory system is indeed being dynamically modulated through hormonal action. The neuropeptide Y (NPY) family peptides, NPY and PYY, play a major role in the regulation of satiety when expressed by gut cells and/or in CNS tissues. Currently, PYY is being viewed as a candidate treatment for obesity and has been through clinical trials because of its ability to reduce food intake. However, unfortunately, high doses of PYY given systemically also cause conditioned taste aversion (CTA). Our data suggesting that PYY introduced into the oral cavity, while leading to significant weight loss in animal models, does not induce CTA, reintroduces PYY as a putative treatment for obesity. The presence of these NPY family peptides in the oral cavity, along with the expression of their cognate receptor(s) in gustatory tissues suggests that they may be influencing ingestive behavior by affecting the functioning of the peripheral gustatory system. Using a combination of genetic and pharmacological models, the proposed studies focus on investigating the general hypothesis that taste-related behavior can be modulated by metabolic hormones such as PYY and/or NPY.

PROJECT SUMMARY Building on the NIDCD Strategic Plan (2017-2022), we propose an interdisciplinary two-day conference to identify high-yield research investment opportunities on smell and taste disorders, with a specific focus on gene therapy and stem cell treatments. Our objectives are to (1) increase collaboration across fields by inviting scientists addressing similar topics in both the chemical senses and other tissues or systems, (2) produce a peer-reviewed, group-consensus recommendation for next steps in research, and (3) communicate conference findings to scientists, clinicians, and patients. Ensuring diversity among attendees is a priority with accommodations offered as needed for people with special needs. The conference will be face-to-face with time for small group discussion. We will also release edited videos of the outreach content.

ABSTRACT The overall goal of this T32 Institutional National Research Science Award proposal is to support predoctoral training in chemical senses research through the Training Program in Chemosensory Science (TPCS) at the University of Florida. The study of the chemical senses (smell, taste, chemesthesis and internal chemosensing) has broad impacts on human health, including: the effects of smell or taste impairments (e.g., anosmia, phantom tastes, etc.) on eating, nutrition, safety, interpersonal relationships and the incidence of depression; the engagement of normal smell and taste to promote healthy eating; the contribution of maladaptive chemosensory behaviors to overconsumption and its related diseases (e.g., diabetes, hypertension, etc.); and the control of disease vectors and parasites through disruption of host seeking or reproduction. Unfortunately, chemosensory scientists with the appropriate methodological expertise and requisite knowledge in this multidisciplinary field continue to be in short supply. This need creates a strong impetus for building an integrative predoctoral training program in chemosensory science. The chemosensory research community at the University of Florida ? highly diverse in research questions and methodologies, but organized and integrated through the UF Center for Smell and Taste ? is uniquely positioned to lead this program.The TPCS has four Specific Aims: (1) To conduct a successful program of predoctoral training in chemosensory science composed of a didactic curriculum, a series of chemical senses research-focused discussions, a mentored research experience, internships in non-academic settings, a patient outreach experience, and professional development; (2) To link predoctoral trainees with strong research mentors and a multidisciplinary committee of experienced investigators; (3) To recruit talented and diverse trainees from national and local pools of eligible candidates; and (4) To evaluate the program in terms of educational objectives tailored to the pre-doctoral program. This application seeks five years' funding for the TPCS to eventually support four predoctoral students per year (two in each of two years of training support per trainee) with the aim of producing independent scientists capable of making significant contributions to the science of smell, taste and chemesthesis. At the conclusion of the period of support, a diverse group of predoctoral trainees will have been mentored and taught the advanced methods, fundamental knowledge, and multidisciplinary approaches necessary to further chemosensory research. Additionally, our unique internship component of training will provide our trainees with substantive exposure to alternative careers in chemosensory science. Together, this program provides a comprehensive training in chemosensory science that will effectively prepare our trainees for the wealth of chemosensory science careers available.

The COVID-19 pandemic is the most devastating infectious disease outbreak in a century, particularly in underserved and minoritized communities. In 2020 alone, it will cost a million lives. It continues to wreak economic havoc worldwide. Therefore, it is critical to develop new tools that can mitigate the spread of SARS- CoV-2, the virus that causes COVID-19. Rapid screening tools can identify potentially infected individuals who can then be isolated/quarantined from the uninfected and directed towards further testing and treatment. Unfortunately, definitive viral testing for SARS-CoV-2 has proven difficult to implement in many countries, including the US, due to technical, financial and governmental hurdles to universal access and timely processing. Symptom-based screening offers a valuable, albeit imperfect, complement to viral testing that can help identify many individuals with the disease for isolation as well as treatment. A major challenge with symptomatic testing is that COVID-19 is highly protean: the heterogeneity of symptoms means no single symptom or constellation of symptoms is definitive diagnostically. Still, there is growing evidence that sudden partial or complete olfactory loss ? even more than other symptoms such as fever or dry cough ? is the single best predictor of COVID-19. In this proposal, we will develop and implement objective, self-administered smell tests for the purpose of identifying individuals with COVID-19 prior to, or in the absence of, viral testing, as well as for use in population-level surveillance of COVID-19 spread. Several kinds of objective tests have been used in clinical or laboratory settings to assess an individual's olfactory ability, including those that test the ability to identify or discriminate odors as well as procedures to determine the lowest concentration an individual can reliably perceive (i.e., odor detection threshold). Each approach has technical and logistical advantages and disadvantages, and each captures different aspects of olfactory dysfunction. Regarding COVID-19, it is unknown what type of measure has the highest specificity or sensitivity. In Aim 1, we will use self-administered objective testing of odor identification and odor detection threshold in SARS-CoV-2-tested individuals to determine which olfactory measure is the best predictor of COVID-19. In Aim 2, we will use objective smell testing to assess whether population monitoring of olfactory loss in university, municipal or other community settings can serve as a sentinel of COVID-19 community spread. Together, our studies will provide a rapid, remote-friendly, cost-effective, scalable, non-intrusive method to screen for COVID-19 at the individual level and to assess prevalence in communities, especially those that have been traditionally underserved by the health care system and public health infrastructure.