Robert F. Margolskee - US grants

DESCRIPTION (Adapted from the Investigator's Abstract): The long term objective is to identify and characterize the various components of taste transduction pathways to understand how they function in the taste cell. Of particular importance are taste cell guanine nucleotide binding regulatory proteins (G proteins) and the seven transmembrane helix receptors that couple to them. G proteins regulate effector enzymes such as phosphodiesterase and phospholipase to effect taste cell changes in intracellular second messengers (e.g., cAMP, cGMP, IP3, Ca2+). Gustducin is a taste specific G protein closely related to the transducins (the photoreceptor G proteins). Recently, it has been shown that rod transducin is also expressed in taste cells. These findings suggest that taste transduction may have some similarities to phototransduction. The specific goals of the proposal are the following: 1) to characterize gustducin's role in taste transduction using biochemical methods; 2) to purify, physically characterize and molecularly clone a recently identified taste specific phosphodiesterase (PDE); 3) to characterize the gustducin-PDE interaction using peptides and mutated gustducin and transducin proteins; 4) to characterize gustducin's role in taste transduction using behavioral and electrophysiological analysis of transgenic mice. Preliminary data indicate significant differences between gustducin knock out and wild type mice in their behavioral and electrophysiological responses to both sweet and bitter compounds; 5) the murine gustducin promoter will be used to express gustducin and transducin as transgenes in the taste cells of gustducin knock out mice. If transducin rescues knock out mice, it will indicate that gustducin and transducin are interchangeable in taste transduction; 6) transgenic mice will be made that express mutant gustducin deficient in its interaction with PDE. These transgenic animals will be tested behaviorally and electrophysiologically to determine if mutant gustducin also leads to altered bitter and sweet responses. Diverse methods will be required to address these goals: biochemical, transgenic, behavioral and electrophysiological techniques will be used. the results of these studies will provide significant new insights into the molecular mechanisms underlying taste transduction. Gustatory and metabolic disorders such as malgeusia, dysgeusia and cachexia frequently occur in conjunction with several types of cancer. The knowledge gained from this proposal may further our understanding of the molecular bases of taste disorders and eventually lead to effective intervention.

DESCRIPTION: Biochemical and physiological studies of the mechanisms underlyi taste transduction suggest that bitter and sweet stimuli are transduced by G-protein-coupled receptors. Five different G proteins are expressed in taste buds (which contain both receptor and non-receptor cell types), and one G-protein, gustducin, is expressed only in taste receptor cells. The responses bitter and sweet stimuli were suppressed in gustducin-knockout mice, although not completely, suggesting that gustducin has some role in these responses. T sequence of gustducin is similar to that of transducin, the photoreceptor G-protein, and both G-proteins can activate a phosphodiesterase from taste tissu however, existing data suggest that this action of gustducin is unlikely to be the primary transduction pathway for either bitter or sweet stimuli. The role of the gustducin-mediated pathways in taste cells remain uncertain. In additi and despite considerable effort, no taste receptors that couple through gustducin or any other G-protein have been cloned; however, bovine rhodopsin which normally couples through transducin, will activate gustducin. The goal of this research application is to generate transgenic mice that express bovine rod opsin in their gustducin-expressing taste receptor cells. Using these engineered taste cells, the taste transduction pathways that invol gustducin will be studied. To do this, methods would be employed that are similar to those used earlier by the applicant to express alpha-galactosidase and Green Fluorescent Protein in the gustducin lineage of taste cells. Taste buds from the transgenic animals would be isolated and incubated in the dark with 11-cis-retinal to regenerate rhodopsin. Then, bright light would be used as a stimulus to activate the receptor. With sufficient light intensity, it i expected that a gustducin-mediated response could be elicited which is wholly dependent upon the endogenous elements in the taste transduction pathway downstream of the receptor. Electrophysiological studies would then be conducted to identify the membrane conductance changes elicited as a consequen of activating this pathway. The strategy of generating the transgenic animals should not discriminate between sweet-responsive and bitter-responsive taste cell phenotypes containin gustducin, so that all the sweet- and bitter-responsive cell types should be represented in the opsin-expressing population. To distinguish between the effects mediated by the gustducin transduction pathways in the two phenotypes, it will be necessary first to identify the gustducin/opsin-containing cells. This will be done by co-expressing Green Fluorescent Protein with bovine opsin to provide a visible marker. Second, the normal phenotypes of cells can be assessed by testing with sweeteners or the very bitter denatonium. Finally, t gustducin-mediated portion of the normal response could be tested by stimulati rhodopsin with light.

DESCRIPTION: The long term objective of this research program is to identify and characterize the various components of taste transduction pathways to understand how they function in the taste cell. Of particular importance are taste cell guanine nucleotide binding regulatory proteins (G proteins) and the seven transmembrane helix receptors that couple to them. G proteins regulate effector enzymes such as phosphodiesterase and phospholipase to effect taste cell changes in intracellular second messengers (e.g. cAMP, cGMP, IP3, Ca++). Gustducin is a taste specific G protein closely related to the transducins. Recently, it has been shown that rod transducin is also expressed in taste cells. These findings suggest that taste transduction may have some similarities to phototransduction. The specific goals of this proposal are the following: 1. To solubilize, reconstitute into lipid vesicles and physically characterize a recently identified apparent taste receptor that is responsive to denatonium. 2. To characterize the interaction of the presumptive denatoniu receptor with gustducin using peptide competition assays and directed mutagenesis of gustducin. 3. To chromatographically purify and molecularly clone this apparent receptor. 4. To characterize the gustducin-denatonium receptor interaction using behavioral and electrophysiological analysis of transgenic mice. Preliminary data demonstrate significant differences between gustducin knock out and wild type mice in their behavioral and electrophysiological responses to both sweet and bitter compounds. Additional preliminary data demonstrate that an 8.4 kb murine gustducin promoter driving expression of a gustducin cDNA transgene corrects the behavioral and electrophysiological deficits of gustducin knock out mice. 5. Transgenic mice will be made that express mutant forms of gustducin deficient in receptor interaction or with increased GDP/GTP exchange. 6. These transgenic animals will be tested behaviorally and electrophysiologically to determine if alterin the gustducin-receptor interaction or guanine nucleotide exchange affects bitter and sweet responses. Diverse methods will be required to address these goals: biochemical, transgenic, behavioral and electrophysiological techniques will be used. The results of these studies will provide significant new insights into the molecular mechanisms underlying bitter and sweet taste transduction. Gustatory and metabolic disorders such as malgeusia, dysgeusia and cachexia frequently occur in conjunction with several types of cancer. The knowledge gained from this proposal could further enhance our understanding of the molecular bases of taste disorders and may lead to effective intervention.

DESCRIPTION (provided by applicant): Funds are requested to assist in the 2001 Gordon Research Conference on the Chemical Senses, July 1-6, Salve Regina University in Newport, RI. Financial support from the NIDCD will be used to defray partially the travel and meeting costs incurred by the invited speakers, discussants, and session organizers. In addition, registration and travel scholarships are requested for young investiga-tors (postdocs and students) with preference given to minority applicants. The meeting will consist of a plenary lecture by Craig Venter (Biology in the Postgenomic Millennium), a workshop on bio-informatics and genomics, and seven focused sessions highlighting the future of chemosensory research. The theme and title of the conference, Chemical Senses in the Postgenomic Era, is particularly timely given the recent completion of the DNA sequences of the genomes of C. elegans, Drosophila and human. Topics chosen are undergoing rapid technological and conceptual growth. All of the invited speakers and discussion leaders are experts in the field of chemoreception or genomics, or are from ancillary fields. In contrast to other meetings involving researchers of the Chemical Senses, such as the annual meeting of the Association for Chemoreception Sciences, the upcoming Gordon Research Conference on the Chemical Senses will be restricted in both scope and attendance. This feature along with the unconventional meeting format is designed to promote uninhibited and penetrating discussion and to generate new insights and directions for future research. The need for a specialized national meeting in the Chemical Senses with emphasis on post-genomic approaches to this field reflects the rapid evolution of knowledge within both genomics and the Chemical Senses and a corresponding growth in the number of talented researchers devoting themselves to these problems. Our aim is to have an open forum, offered uniquely by the Gordon Research Conference format, whereby experts in the field can share their most recent findings, thoughts, and speculations. Consequently, this should further progress in chemosensory research and aid the overall mission of the NIDCD.

DESCRIPTION (provided by applicant): The long-term objective of our research program is to understand how the vertebrate gustatory system transduces signals and encodes information. Of particular importance are taste cell guanine nucleotide binding regulatory proteins (G proteins) and the seven transmembrane helix receptors that couple to them. G proteins regulate effector enzymes such as phosphodiesterase (PDE) and phospholipase C (PLC) to effect taste cell changes in intracellular second messengers. We have used molecular cloning to identify a number of the key components of taste transduction pathways, including Alpha-gustducin, a taste specific G protein Alpha -subunit closely related to the Alpha-transducins. The scope of this Competing Continuation encompasses the roles in taste transduction and coding of heterotrimeric gustducin?s component subunits and the downstream effector enzymes to which they couple. To elucidate the specific roles in taste transduction and coding of Alpha-gustducin, BetaGamma-gustducin, Ggamma13, taste-expressed PDEs and PLCBeta2 we will use a multidisciplinary approach applying biochemical, transgenic, behavioral, and electrophysiological techniques. The Experimental Goals of the proposal are the following. 1. To determine which PDEs present in taste tissue can be activated by alpha-gustducin. 2. To determine if these PDEs are co-expressed with alpha-gustducin in taste cells. 3. To determine if these PDEs can be activated by any other G protein alpha-subunits co-expressed with them in taste cells. 4. To generate alpha-gustducin mutants selectively deficient in the ability to activate the taste PDEs. 5. To generate transgenic mice expressing such an alpha-gustducin mutant. 6. To biochemically characterize taste transduction responses of the alpha-gustducin mutant mice. 7. To behaviorally characterize the alpha-gustducin mutant mice. 8. To electrophysiologically characterize the alpha-gustducin mutant mice. 9. To generate transgenic mice lacking signals mediated by gustducin?s beta, gamma-subunits. 10. To biochemically characterize taste transduction responses of the beta gamma-gustducin deficient mice. 11. To behaviorally characterize the beta, gamma-gustducin deficient mice. 12. To electrophysiologically characterize the beta, gamma-gustducin deficient mice. The results of these studies will provide significant new insights into the molecular mechanisms underlying taste transduction and coding. Gustatory and metabolic disorders such as malgeusia, dysgeusia and cachexia frequently occur in conjunction with several types of cancer. The knowledge gained from this proposal should further our understanding of the molecular bases of the taste disorders and may lead to effective intervention.

[unreadable] DESCRIPTION (provided by applicant): The long-term objective of our research program is to understand how the vertebrate gustatory system transduces signals and encodes information. Of particular importance are the G protein-coupled receptors (GPCRs) and their coupled G proteins. During the past decade we have identified and characterized several taste transduction elements, including taste receptors (Tlr3), G proteins ((-gustducin, (-transducin, G(13) and ion channels (Trpm5). The scope of this Competing Continuation encompasses the roles in taste transduction and coding of Tlr taste receptors, the G proteins co-expressed with and coupled to these receptors, and the transient receptor potential (Trp) channels expressed in Tlr-positive taste cells. We will use a multidisciplinary approach applying molecular biological, transgenic, behavioral and electrophysiological techniques to achieve the following Specific Aims. 1. To determine if taste cells express Tlr4. 2. To determine which Tlr receptors are co-expressed with Tlr4. 3. To determine which G protein subunits are co-expressed with Tlr receptors. 4. To determine which Trp channels are co-expressed with Tlr receptors. 5. To heterologously express Tlr receptors. 6. To use Caimaging to monitor responses of cells expressing the Tlr receptors. 7. To generate single, double and triple knockout mice lacking Tlr receptors. 8. To behaviorally characterize the Tlr knockout mice. 9. To electrophysiologically characterize the Tlr knockout mice. 10. To generate knockout mice lacking Trpm5. 11. To generate transgenic mice expressing Trpm5 in Tlr2- and Tlr3-positive taste cells. 12. To behaviorally characterize the Trpm5 transgenic mice. 13. To electrophysiologically characterize the Trpm5 transgenic mice. The results of these studies will provide significant new insights into the molecular mechanisms underlying taste transduction and coding. The Tlr receptors are involved in the detection of sugars and sweeteners. A molecular knowledge of their mode of function may lead to the development of novel effective non-caloric sweeteners that may be useful in weight control programs to limit obesity and obesity-related diseases. Gustatory and metabolic disorders such as malgeusia, dysgeusia and cachexia frequently occur in conjunction with several types of cancer. The knowledge gained from this proposal should further our understanding of the molecular bases of taste disorders and may lead to effective intervention. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The long-range goal of our research program is to understand the molecular events underlying function of the heterodimeric sweet taste receptor (T1R2+T1R3). The sweet receptor is a remarkably broadly acting receptor, capable of responding to native and artificial sweeteners. In vivo and in vitro studies suggest that this single heterodimeric receptor is the primary or only sweet taste receptor. We are interested in how so many chemically diverse ligands can bind to the sweet receptor, how binding at different sites leads to receptor activation, and how the domains of each T1R monomer contribute to binding, activation and signal transduction. To address these goals we have developed ligand binding and activity assays, and used these techniques in concert with mutagenesis and molecular modeling to begin to understand this complex receptor. The present proposal applies these several techniques to examine how the small molecule-binding site of T1R2 interacts with aspartame, neotame and alitame (so-called dipeptide sweeteners). This "canonical" binding site is found within the "venus fly trap module" (VFTM) of T1R2. In Aim 1 we will use the differential sensitivity of the human and mouse sweet receptors to dipeptide sweeteners, along with heterologous expression assays, to identify key residues within the VFTM of T1R2 involved in the interactions with dipeptide sweeteners. In Aim 2 we will use directed mutagenesis of T1R2, heterologous assays and molecular modeling to physically and chemically characterize the interaction of dipeptide sweeteners with the VFTM of T1R2. In Aim 3 we will use spectroscopic and calorimetric techniques to monitor binding of dipeptide sweeteners to the expressed VFTM of T1R2 and T1R2 mutants. There is today in the affluent countries of the world an epidemic of obesity, insulin-resistant diabetes and diet-related disorders. In our evolutionary past a strong drive to consume high-carbohydrate/energy-rich foods was advantageous for survival. Today, our more sedentary lives and the ready availability of food makes this sweet-seeking behavior a liability that may contribute significantly to obesity. Sweet taste perception mediated by the heterodimeric sweet taste receptor undoubtedly contributes to sweet-seeking behavior and food consumption. The studies in this proposal will enhance our understanding at the molecular level of sweet receptor function with the hope of future means to control our sweet cravings and the attendant diseases of over-consumption. [unreadable] [unreadable] [unreadable]

Description (provided by applicant): During the past few years my laboratory has explored the involvement of taste signaling elements in gastrointestinal (GI) chemosensation. In particular, how macronutrients in the gut lumen are sensed and in turn regulate hormone release from specific enteroendocrine cells of the gut. We have determined that the very same signaling elements that underlie taste detection/transduction also are expressed in specific subtypes of enteroendocrine cells, so-called taste cells of the gut. Moreover, these signaling proteins function in the gut cells to sense, in effect to taste, the dietary contents within the lumen of the gut. Chemosensory responses of these enteroendocrine cells include hormone release to promote the gut's digestive and metabolic functions. A striking example of the importance of taste signaling elements to GI chemosensation comes from the observation that 1-gustducin knockout mice do not secrete GLP-1 (Glucagon-Like Peptide 1) from their enteroendocrine cells in response to infusion of glucose into the gut lumen - because of this defect these mice have dysfunctional regulation of their plasma insulin and glucose levels, leading them to a prediabetic condition. Quite recently we have observed that a number of hormones and signaling elements typically found in enteroendocrine cells of the gut are present also in a subset of taste cells, which we're calling lingual endocrine cells or endocrine taste cells. We hypothesize that in response to tastants these taste cells release GLP-1 and other hormones that have local (paracrine) and/or systemic effects relevant to taste, digestion and satiety. Further, we hypothesize that taste signaling mediated by gustducin, T1R3, Trpm5 and other taste transduction elements contributes importantly to the endocrine and paracrine responses of these lingual endocrine cells. Our preliminary data show that GLP-1, glucagon and PYY (peptide YY) are expressed in endocrine taste cells. In addition, we have found that glucose and sweeteners elicit release of GLP-1 from these taste cells. The experiments proposed here aim to further characterize the functions of these lingual endocrine cells. We have combined molecular, cellular, transgenic, physiological and pharmacological methods to determine: 1. which gut signaling molecules are expressed in lingual endocrine cells; 2. which hormones are released from these endocrine taste cells in response to specific tastants; 3. which taste signaling elements are involved in tastant- elicited hormone release from endocrine taste cells; and 4. what are the local or systemic effects of endocrine taste cell-released hormones. This multidisciplinary approach has promise for providing significant new insights into the nature of cephalic phase responses regulating taste cell release of GLP-1 and other hormones to regulate gut functions. This proposal has medical relevance to appetite, satiety, diabetes and obesity. The same signaling elements that underlie taste detection/transduction also are expressed in gut enteroendocrine cells, where they function to sense the dietary contents within the lumen of the gut. A number of hormones typically found in enteroendocrine cells of the gut are present also in a subset of taste cells, called lingual endocrine cells or endocrine taste cells. The experiments proposed to identify and functionally characterize these lingual endocrine cells have medical relevance to appetite, satiety, diabetes and obesity.

DESCRIPTION (provided by applicant): The Monell Chemical Senses Center is a unique multidisciplinary institute devoted to investigating the science of the chemical senses. Currently there are 21 participating faculty members representing disciplines ranging from molecular biology and genetics to psychophysics and nutrition and conducting research in both basic and clinical aspects of olfaction and gustation. The Monell Center has enjoyed a successful Interdisciplinary Training Program in the Chemical Senses for over 30 years. The long-term goal of the training program is to provide a pool of scientists well-trained in the chemical senses who are capable of becoming independent scientists. Trainees from a wide variety of scientific backgrounds both within and outside of the chemical senses area are recruited to the program. The Postdoctoral Training Program consists of didactic courses, research training and research experience, grant writing as well as training in the ethical principals of scientific research. Trainees are assigned a mentoring team to ensure completion of required training components and obtainment of scientific goals such as publications and grant submissions. This proposal requests funds to defray the costs of training four postdoctoral fellows per year. PUBLIC HEALTH RELEVANCE: The Monell Chemical Senses Center is a unique multi-disciplinary research institute devoted to the study of the chemical senses. The goal of this proposal is to provide interdisciplinary post-doctoral training in the chemical senses for trainees coming from a wide range of scientific disciplines. This is achieved by providing the tools necessary for young scientists to gain independence: a strong scientific environment, committed faculty and didactic course work in appropriate areas.

DESCRIPTION (provided by applicant): We have determined that gustducin, T1R3 and several other signaling elements that underlie taste detection/transduction also are expressed in L and K subtypes of enteroendocrine cells. One function of gustducin and T1R3 in L cells is to sense glucose or sweeteners within the lumen of the gut. 1-Gustducin knockout mice do not secrete GLP-1 from their enteroendocrine L cells in response to infusion of glucose into the gut lumen. Because of this defect these mice have dysfunctional regulation of their plasma insulin and glucose levels. Studies with minced duodenal tissue or isolated duodenal villi from 1-gustducin knockout mice show that the defect in GLP-1 secretion is independent of innervation. Studies with human and mouse enteroendocrine cell lines and antisense blockade of gustducin or pharmacological inhibition of T1R3 indicate that both gustducin and T1R3 are required for enteroendocrine cell release of GLP-1, suggesting that the defect in 1-gustducin knockout mice in vivo is at the level of glucose sensation. The experiments proposed here aim to further characterize the functions of gustducin, T1R3 and other taste signaling elements in enteroendocrine cells. We have combined molecular, cellular, transgenic, physiological and pharmacological methods to: 1. Determine which key taste signaling molecules are expressed in which enteroendocrine cells; 2. Determine if hormone release from enteroendocrine cells depends on their expression of taste signaling elements and elicits upregulation of sugar and fatty acid transporters in enterocytes; 3. Determine if pharmacologic or genetic block of enteroendocrine cell-expressed taste signaling elements alters hormone release from enteroendocrine cells; 4. Generate conditional knockout mice selectively lacking 1-gustducin, T1r3 or Trpm5 in gut; 5. Determine if conditional knockout mice lacking 1- gustducin, T1r3 or Trpm5 in gut are resistant to diet-induced obesity, show defective 2 cell function on a high fat diet, and exhibit increased energy expenditure. We will be testing the following key hypotheses: 1. Specific types of enteroendocrine cell type express multiple taste signaling elements; 2. Macronutrients and tastants elicit release of GLP-1 and other hormones from enteroendocrine cell by activating gustducin and taste receptors expressed in subtypes of enteroendocrine cells; 3. Conditional Knockout mice lacking enteroendocrine cell-expressed taste signaling elements will have defects in regulating their plasma levels of GLP-1, GIP and other hormones - leading to: (a) failure to upregulate certain enterocyte transporters, (b) dysregulation of glucose homeostasis, (c) resistance to diet-induced obesity, (d) disruption of 2 cell functions, (e) increased energy expenditure. This multidisciplinary approach has promise for providing significant new insights into the nature of enteroendocrine cell function. In particular, how enteroendocrine cells sense macronutrients (e.g. glucose and sugars) and tastants (e.g. artificial sweeteners) in the gut lumen, and how this leads to stimulation of release of GLP-1 and other hormones to regulate gut functions. This proposal has medical relevance to appetite, satiety, obesity, and diabetes.

DESCRIPTION (provided by applicant): Modulating the function of G protein coupled receptors (GPCRs) is a common means by which the sensitivity of signaling pathways can be increased or decreased as circumstances dictate. Different accessory enhancing proteins can affect GPCR responsiveness in several ways: regulate expression, target subcellular localization, promote proper folding, or enhance association with G proteins. Olfactory receptors, rhodopsin and taste receptors are amongst the sensory GPCRs for which one or more accessory enhancing proteins have been found to modulate the receptor's response function - particularly important for receptors that must function to detect sensory stimuli over several orders of magnitude. Meyerhof and colleagues and we have found multiple RTPs (Receptor Transporting Proteins) and REEPs (Receptor Expression Enhancing Proteins) to be present in taste tissue. Contrary to the results of Meyerhof's group we observed that REEP2 is selectively expressed in taste cells and enhances the function of both sweet and bitter taste receptors. The experiments proposed here functionally characterize the roles of RTPs and REEPs, particularly REEP2, in promoting taste receptor function. In this proposal we combine molecular, cellular, transgenic, physiological and behavioral methods to determine: 1. which receptor enhancing proteins are expressed in which taste cells; 2. which receptor enhancing proteins interact with which taste receptors and by which sites; 3. how REEP2 and other REEPs/RTPs enhance sweet receptor function; 4. if REEP2 and and/or other REEPs/RTPs are involved in taste receptor function in vivo. This multidisciplinary approach has promise for providing significant new insights into the function and regulation of bitter, sweet and amino acid taste receptors within taste cells. This proposal has medical relevance to gustatory function, appetite, satiety, diabetes and obesity. PUBLIC HEALTH RELEVANCE: In many sensory systems accessory proteins regulate or enhance the function of the sensory receptor. We have found that one such receptor enhancing protein, REEP2, is selectively expressed in taste cells and enhances responses from bitter and sweet taste receptors but not from non-gustatory receptors. The experiments proposed to determine how REEP2 and other REEPs and RTPs interact with taste receptors and if this occurs in vivo have medical relevance to gustatory function, appetite, satiety, diabetes and obesity.

The Monell Chemical Senses Center, founded in 1968, is the world's first and only multidisciplinary institute devoted entirely to basic research in the chemical senses and produces extensive research within the NIDCD mission area. Two of Monell's greatest strengths are the breadth of approaches brought to bear on problems in the chemical senses, and the effective interdisciplinary collaborations that are fostered at the Monell Center. In this P30 grant application for a Core Center in the chemical senses, four Research Cores with distinct areas of technical expertise highly relevant to the chemical senses are proposed: 1. Histology, 2. Genotyping, 3. Molecular Biology, and 4. Phenotyping. In addition, an Administrative Core will support the Research Cores in their mission to enhance the research of users in our Research Base of thirteen R01 grants in the NIDCD mission area. Dr. Liquan Huang will lead the Histology Core, building on his expertise in the visualization of proteins in taste receptor cells. The Genotyping Core will be led by Dr. Danielle Reed, who has over twenty years of training and experience with genotyping and gene mapping. Dr. Bedrich Mosinger, an expert in molecular biology and in constructing transgenic mouse models, is the Director of the Molecular Biology Core. Dr. Alexander Bachmanov will run the Phenotyping Core; he is well-known for his ability to phenotype mice and rats for traits relevant to the chemical senses. The Director of the Administrative Core is Dr. Robert Margolskee, who through his expertise in the research areas of all four Research Cores and his administrative experience is uniquely qualified to serve as PI of this Core Center grant. Together these Cores will serve the Research Base at Monell and by contributing to innovative research in the chemical senses further the mission of NIDCD to understand taste, smell and the common chemical sense in order to improve human health and well-being.

The Administrative Shell will coordinate and manage all activities of the PSO Core Center for the Chemical Senses. The PI of the Core Center, Dr. Robert Margolskee. and the Administrative Coordinator. Ms. Lee, will coordinate operations of the Core Center through the Administrative Shell. Specific administrative functions of the Shell include coordinating between the Core Center's Research Cores and Research Base and seeing that the Research Cores are fully utilized, that priority is granted to Research Base users, that fair use of Cores is followed, and that the Core's procedures for dispute resolution are followed. Administrative Shell personnel also are responsible for tracking Core Center expenses, monitoring chargeback costs for each Core, booking lectures and training sessions given by the Research Core Directors, scheduling meetings of the Core Center's Internal and External Advisory Boards, and assisting with making travel arrangements for the external advisors. The Monell Chemical Senses Center was founded in 1968 on the premise that basic and clinical research into the health-related impact of the chemical senses would be facilitated by having a center of excellence devoted exclusively to these topics. As a 501(C)(3) organization, Monell is headed by a Board of Directors who are legally and fiscally responsible for its operations. The Board is headed by Dr. Dwight Riskey, former Senior Vice President of PepsiCo. Among its members are two University of Pennsylvania trustees and one senior member of the University of Pennsylvania medical faculty, the President of the Monell Foundation, the CEO of AAAS, a senior executive of the Pew Charitable Trusts, and Dr. Gary Beauchamp. the Director of Monell since 1990. Monell has several advisory groups, the most important of which is the International Advisory Committee made up of distinguished individuals from academia and industry. In addition to overseeing longterm fiscal and research activities at Monell, the Board appoints the Director, who is responsible for day-to-day operations at Monell. The Director is also involved in long-term strategic planning, faculty appointments, and resource allocation. The PSO Core Center for the Chemical Senses is a natural extension of Monell's 42-year mission. The organizational structure of Monell and how the Core Center fits in is shown in Figure 1. The Shell promotes the mission of our Core Center to provide efficient technical services and educate our users in new technologies. For each Research Core, their laboratories and the offices of their Directors are within the same building, often on the same floor, and sometimes in adjacent space. The physical distances between Cores, which are in two adjacent, connected buildings (e.g.. Core 1. Histology in Monell East and Core 2. Genotyping in Monell West), are modest and can be covered in 5 minutes or less. Frequent (daily) conversations occur among all Core Directors. The Administrative Shell's role in the PSO Core Center is key to facilitate these interactions, yet the size and cost of the Administrative Shell are modest, taking up only -6% of the total direct costs of the PSO Core Center. The P30 PI and Core Center Director overseeing the Administrative Shell is Dr. Robert Margolskee. Dr. Margolskee is a world-renowned expert on the molecular mechanisms of taste signaling. His group discovered the first taste transduction protein, gustducin, as well as additional taste signaling proteins: the G-protein subunit Gyl 3, the sweet and umami taste receptor subunit Tl rS, and the Ca2+-activated cation channel TrpmS. His lab was the first to generate and utilize transgenic models in the chemical senses with studies of gustducin-null mice. His group has also generated and characterized knockout mice lacking transducin, TIrS, and Trpm5. His several seminal contributions to the taste field have been published in high-impact journals, including Nature, Science, Nature Genetics, and Proceedings of the National Academy of Sciences of the USA. Having trained in molecular genetics, molecular neurobiology, and medicine, his research program is characterized by an interdisciplinary approach utilizing biochemistry, molecular biology, transgenic mouse models, behavior, and physiology. During the past few years he has extended his studies to investigate the roles of taste-signaling proteins in endocrine responses of extra-oral tissues. His group recently demonstrated that gustducin, TIrS, and other taste elements are expressed in intestinal and pancreatic endocrine cells, where they are involved in detecting sugars and sweeteners and eliciting hormone release. Dr. Margolskee joined Monell in July of 2009 as a Member, Monell Center's highest faculty level. He is also Director of Monell's TS2 Postdoctoral Training Program. For the prior 1S years he was on the faculty of the Mount Sinai School of Medicine in the Departments of Neuroscience, Pharmacology, and Physiology and Biophysics. For eight of those years he was also an Investigator with the Howard Hughes Medical Institute. During his 23 years as a PI, Dr. Margolskee has trained 40 pre- and postdoctoral fellows. Many of his trainees have gone on to establish themselves in academia or industry. Dr. Margolskee currently holds three ROIs, all of which are in the Research Base, and two industry-funded research grants. Dr. Margolskee has significant administrative experience: in addition to Directing Monell's T32 Postdoctoral Training Program, he has served as Co-Director of Mount Sinai's Graduate Training Area in Mechanisms of Disease and Therapy, Founder and Acting President of Linguagen/Redpoint Bio (a biotech company). Program Chair for the Association for Chemoreception Sciences annual meeting, and Co-Chair of the Gordon Conference in the Chemical Senses. Dr. Margolskee will oversee the PSO Core Center and coordinate Core personnel. He is well suited for this role based on his scientific expertise and administrative experience. His research in taste signaling and the roles of taste signaling proteins in extra-oral sites is central to the mission area of NIDCD. Dr. Margolskee has many years of direct experience and has acquired skills and knowledge in each of the technical areas covered by the four Research Cores: Histology, Genotyping, Molecular Biology, and Phenotyping. Specific examples from his work include the creation and characterization of mice null for the taste cell signaling molecules gustducin, transducin, Trpm5, and T1r3. His group has created gene constructs for conventional and conditional null mice, made transgenic mouse models, genotyped mice, localized proteins and mRNAs to subsets of taste receptors cells, done molecular biological analyses of gene products in cell culture and of genes in vivo, and carried out two-bottle preference tests, gustometer tests, gustatory nerve recording, and surgical manipulation of gustatory and gut physiology. Dr. Margolskee and all four core directors (Drs. Huang, Reed, Mosinger, and Bachmanov) talk many times a day concerning scientific and administrative matters. Drs. Huang, Reed, and Bachmanov have adjacent offices. The frequent collaboration common among Dr. Margolskee and the Research Core Directors are exemplified by the multiple co-authored publications; for instance. Dr. Margolskee has co-authored papers with each of the four Core Directors, Drs. Reed and Bachmanov have jointly authored 19 peer-reviewed papers, and Drs. Margolskee and Mosinger have jointly authored 17 papers. Because of the physical proximity and the long-standing collaborations of the Core Directors, questions or problems about Center management generally can be resolved quickly and amicably.

? DESCRIPTION (provided by applicant): The sweet taste receptor protein T1r2+T1r3, encoded by the Tas1r2 and Tas1r3 genes, mediates taste responses to non-caloric sweeteners. However, Tas1r3 knockout mice generated in our group retain taste nerve responses to glucose and maltose. Glucose transporters (GLUTs) and metabolic sensors (KATP) expressed selectively in Tas1r3-positive taste cells may underlie taste responses of Tas1r3 knockout mice to sugars. Tas1r3-positive taste cells also express two intestinal type enzymes, sucrase-isomaltase and maltase-glucoamylase, that hydrolyze sucrose and maltose. The general goals of this grant are to elucidate the mechanisms underlying taste detection of sugars and to identify transcription factors that control the generation of sweet-responsive taste cells. The experiments proposed here use gene knockout mice and enzyme inhibitors to assess the involvement of KATP, sucrase-isomaltase and maltase-glucoamylase in taste responses to sugars. In the first two aims of the proposal we combine transgenic, physiological and behavioral methods to determine: 1. If sugar transporters and KATP metabolic sensors contribute to taste transduction of sugars, and 2. If intestinal disaccharidase enzymes expressed in Tas1r3-positive taste cells contribute to taste transduction of sugars. In the third aim we build on results from bioinformatic analysis of genes expressed in taste cells to: (a) Identify transcription factors selectively expressed in Tas1r3+ taste cells, and (b) Determine if loss of transcription factors selectively expressed in Tas1r3-positive taste cells affects generation and function of these cells. Ultimately, these studies may provide insights into why sugars are preferred over non-caloric sweeteners, lead to the generation of superior non-caloric sweeteners, and provide a means to regulate sweet-responsive taste cells so as to decrease the drive for consuming sugar. This multidisciplinary approach has promise for providing significant new insights into the function and regulation of sweet taste receptor cells. This proposal has medical relevance to gustatory function, appetite, satiety, diabetes and obesity.

? DESCRIPTION (provided by applicant) There is overwhelming evidence that the sweet receptor subunits T1R2 and T1R3 are key to the sense of sweet taste in mice, as well as in humans. Despite the clear importance of T1R receptors to sweet taste, there is evidence that alternative pathways exist for detection or modulation of sweet taste. Commonalities among taste cells of tongue and taste-like endocrine cells of gut and pancreas make it plausible that intestinal-type sugar sensors (e.g., glucose transporters (GLUTs) and sodium-glucose co-transporters (SGLTs)) or pancreatic-type metabolic sensors (ATP-gated KATP channels) might also be present in taste cells and function in sweet sensation of sugars. The main goal of this project is to identify and characterize T1R- independent mechanisms used by human taste receptor cells to sense sugars and calories. We hypothesize that sugar transporters and metabolic sensors underlie T1R-independent sugar sensing in human taste receptor cells. We also hypothesize that metabolic responses of human taste cells contribute to the perception of sweet taste and help impart the stronger preference for nutritive over non-caloric sweeteners. We hypothesize further that metabolism of glucose transported into sweet-responsive human taste cells leads to elevated intracellular ATP that closes the taste cell's KATP channels, depolarizing the cell. We will test these hypotheses using histological and functional studies in cultured human taste receptor cells and taste psychophysical tests in human subjects. Together these studies will determine if sugar transporters and KATP channels are present and active in human taste cells and if they are likely to contribute to sugar sensing and oral reward in human subjects. If this metabolic sensor in the sweet taste system can be stimulated without adding calories, it could provide an effective means to help reduce excess sugar in the US diet, thereby reducing the risk of obesity and other diseases associated with overconsumption of calories.

Project Summary Periodontal disease, which results from bacterial infection and inflammation of the gums and bone that surround and support the teeth, affects nearly half of US adults over 30, with ~9% having severe periodontitis. The hallmark of periodontitis is destruction of alveolar bone, resulting ultimately in extended tooth loss and oral disability. Periodontitis is a major oral health problem, particularly among the elderly, that increases the risk for systemic diseases, including atherosclerosis, rheumatoid arthritis, and diabetes mellitus. The general goals of this grant are to determine the role of newly discovered gingival solitary chemosensory cells (gSCCs) in protecting against periodontitis, to identify the receptors, signaling pathways and effectors involved in innate immunity evoked by gSCC activation, and to identify compounds that activate gSCCs to harness host innate immunity to reduce periodontitis. Periodontitis results from polymicrobial dysbiosis, which perturbs the ecologically balanced oral microbiota, and from disruption of the host innate immunity, which also contributes to the destruction of periodontal tissue. While much is known about how microbes and host immunity contribute to periodontitis, it is still unclear which gingival cells protect against the disease. Recent studies in several types of mucosae have identified taste cell-like SCCs as specialized chemosensitive sentinel cells that detect bacteria and evoke host innate immune responses. Most recently, we have identified gSCCs in the mouse gingival junctional epithelium that is part of the epithelial barrier protecting against bacterial infection of the tooth and surrounding gingiva. gSCCs express bitter taste receptors along with other taste transduction components, respond to bacterial signaling molecules, and trigger intrinsic innate immunity to protect against periodontitis. The experiments of this proposal study the role of gSCCs in gingival/tooth health to determine if gSCCs evoke innate immune responses that prevent overgrowth of oral bacteria in the gingival mucosa (Aim 1); identify the receptors and signaling components of gSCCs and the mechanism by which gSCCs promote gingival release of antimicrobial peptides and inflammatory cytokines (Aim 2); and determine if ?on demand ?activation of the gSCC signaling pathway reduces periodontitis (Aim 3). Understanding the initiating receptors and underlying mechanisms of these responses may lead to new approaches to promote oral health and treat periodontitis.

Abstract Relatedness of Supplement Aim to Parent Grant In the parent grant, we use cultured human taste (HBO) cells, pioneered at Monell by Co-Investigator Hakan Ozdener, to probe the metabolic sweet taste signaling pathway. HBO cells provide a useful model for probing taste signaling in culture, but they have also been shown useful for investigating the pathophysiology of certain neurotrophic viral diseases (e.g. Zika virus; see Ozdener et al., 2020). Using HBO cells to accomplish the Supplement Aim will advance our understanding of the pathogenicity of SARS-CoV-2 and other viruses that adversely affect taste and olfaction. Although many studies have reported taste and olfactory loss in individuals with COVID-19 disease, the underlying mechanisms and cellular effects in taste cells are not well understood. Due to changes in taste function in patients with COVID-19, it will be of particular interest to the parent grant to know if the subset of sweet taste cells is susceptible to infection by SARS-CoV-2.