Hiroaki Matsunami - US grants

[unreadable] DESCRIPTION (provided by applicant): Human's ability to detect chemicals though our sense of smell and taste is essential to maintain the quality of our daily life, from enjoying meals and flowers to detecting fire and spoiled food. Our long-term objective is to understand how chemicals are detected in the peripheral sense organs, how the information is processed in the brain to recognize chemicals, and how the brain directs appropriate behavioral responses. These processes are critical for animals to survive and reproduce. Without functional olfactory and gustatory systems, animals have difficulties in detecting and evaluating food, finding predators, prey, mating partners, and noxious chemicals in the environment. Although mammalian odorant receptors (ORs) were identified over 15 years ago, comprehensive understanding of how different odorant molecules interact with ORs at a molecular level has not been achieved. To address this, it is important to have a more complete understanding of the biosynthesis of ORs and OR ligand specificities. Progress on this front has been slow, largely due to the lack of an efficient system for identifying ligands that activate the ORs. In the current grant period, we have made progress in both the olfactory and gustatory systems. In the olfactory system, we identified RTP1 and RTP2, accessory molecules that enhance the cell-surface expression of ORs, enabling us to functionally express ORs in heterologous cells. Using this approach, we identified a set of active ligands for mammalian ORs. In the gustatory system, we identified PKD1L3 and PKD2L1 as candidate sour taste receptors. The Public Health Relevance: There is not a scientist or perfumer in the world who can view a novel molecular structure and predict how it will smell, largely due to a lack of knowledge about which odorant receptors are activated by a given odor. The goals of the studies proposed here are to deepen our understanding of how odorants are detected by odorant receptors. These studies may help understand olfactory dysfunction and develop new ways to improve the quality of our daily life - from enjoying meals and flowers to detecting fire and spoiled food. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): TRP channels are implicated as necessary signaling components in various sensory systems of diverse animal species. Among TRP related genes, PKD1 and PKD2 are two major causative genes for autosomal dominant polycystic kidney disease. We have recently identified two PKD-like proteins, PKD1L3 and PKD2L1, as candidate mammalian sour receptors functioning in taste reception. PKD1L3 and PKD2L1 are coexpressed in a subset of taste receptor cells distinct from those taste cells having receptors for bitter, sweet or umami (savory) tasting chemicals. We found that PKD1L3 and PKD2L1 form a protein complex and coexpression of PKD1L3 and PKD2L1 is necessary for their functional cell surface expression. Finally, our data indicated that when coexpressed in heterologous cells, PKD1L3 and PKD2L1 are activated by various acids, but not by other classes of taste chemicals, osmolarity, or mechanical flow, suggesting that PKD1L3 and PKD2L1 function as sour taste receptors. Based on these and other findings, we propose the following projects to elucidate the in vivo function of the PKD family members. First, we will generate gene knockout mice for PKD1L3 and PKD2L1. We have completed creating a gene knockout vector for PKD2L1 and we are at the final step in creating a gene knockout vector for PKD1L3. Second, we will start analyzing the function of PKD1L3 and PKD2L1 in sour taste detection. We will analyze the homozygous and heterozygous transgenic mice using histological examinations, gustatory nerve recordings and behavioral taste tests. Third, we will generate PKD1L3 and PKD2L1 double gene knockout mice. Autosomal dominant polycistic kidney disease (ADPKD) is a genetic disorder with two major causative genes, PKD1 and PKD2. Recently we have identified two PKD-like proteins, PKD1L3 and PKD2L1, as acid receptors functioning in taste reception in mammals. The relevance of our studies to the public is that it will shed light on how PKD related proteins function as sensors for extracellular stimuli and may help develop a way to prevent ADPKD from being developed [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The ligand selectivity of the three families of receptors expressed in the vomeronasal organ-V1Rs, V2Rs and FPRs-is largely unknown. To deepen our understanding of the interactions between the vomeronasal receptors and their ligands, we propose to establish systems to identify active ligands for the V2Rs and the FPRs. In addition, we propose to identify which domains of the receptor proteins determine ligand selectivity. To test the hypothesis that specific V2R or FPR vomeronasal receptors are activated by specific set of ligands, three Specific Aims are proposed: Specific Aim 1. Characterize the ligand selectivity of V2Rs. We will establish a heterologous expression system for the V2Rs. We will use this system to identify active ligands of the V2Rs and examine the structural basis of ligand selectivity by V2R family members. Specific Aim 2: Characterize the ligand selectivity of VNO-FPRs. We will establish a heterologous expression system for the VNO-FPRs. We will use this system to identify the active ligands of VNO-FPRs. Specific Aim 3: Characterize the ligand selectivity of VNO receptors using VNO sensory neurons. We will establish a system for measuring the response of dissociated vomeronasal neurons using calcium imaging and then measuring the mRNA expression in the same cells. We will use this method to identify the receptors expressed in the cells activated by a given ligand. The proposed experiments further our long term goal, which is to understand the mechanistic basis for ligand recognition by the vomeronasal receptors. The health relatedness of these studies is that a better understanding of the ligand binding attributes of vomeronasal receptors will offer insights into the general features of ligand recognition by G-protein coupled receptors, a class of receptor that is frequently targeted by drugs. This work will improve our understanding of how G protein-coupled vomeronasal receptors recognize and discriminate specific ligands. This basic knowledge will translate to greater understanding of G protein-coupled receptors, which are major pharmaceutical targets for cardiac, psychiatric, and cancer disease states. An understanding of the interactions between the ligand and receptor provides crucial information needed to develop a wide range of health-related products. PUBLIC HEALTH RELEVANCE: Vomeronasal receptors are thought to play an important role in detecting pheromones, although it is unclear which receptors are activated by which pheromones. Our goal is to deepen our understanding of the interactions between the vomeronasal receptors and their ligands. This work is relevant to improving public health, because a better understanding of the ligand binding attributes of vomeronasal receptors will offer insights into the protein-ligand interactions of G-protein coupled receptors, a class of receptors frequently targeted by pharmaceutical drugs.

DESCRIPTION (provided by applicant): Odor perception in mammals is a complex process mediated by the activation of a family of G- protein coupled receptors (GPCRs) known as odorant receptors (ORs) expressed at the cilia of millions of olfactory sensory neurons (OSNs) lining the olfactory epithelium. The olfactory epithelium is richly innervated by autonomic nerves, including parasympathetic nerve endings which release acetylcholine. We recently discovered that OSNs express the type 3 muscarinic acetylcholine receptor (M3R), another GPCR. However, the molecular mechanisms underlying cholinergic actions on OSNs are not well-understood. We propose experiments to address the importance of modulation of olfaction and to clarify the molecular mechanisms that underlie the effects of acetylcholine receptors on OSN signaling. This work will further our understanding of how information processing occurs during the earliest steps of olfaction. To test the hypothesis that the M3R is important for the regulatio of OR function and olfactory signal transduction, the following Specific Aims are proposed: Aim 1. To test the hypothesis that M3R modulates the function of olfactory sensory neurons Aim 2. To test the hypothesis that M3R is involved in odor-mediated activation of alternative signaling Aim 3. To identify structural domains of M3R and signaling components important for the functional interaction of M3R and OR The proposed experiments further our long term goal, which is to understand the mechanistic basis for regulation of odorant receptor function. The health relatedness of these studies is that a better understanding of the regulatory mechanism of ORs and downstream signal transduction will offer insights into the general features of signaling by GPCRs, the most frequent pharmaceutical targets for cardiac, psychiatric, and cancer diseases. This work will advance our understanding on how G protein-coupled ORs are regulated by another GPCR, M3R, which is activated by acetylcholine, the key neurotransmitter of the parasympathetic nervous system. An understanding of the interactions between the neurotransmitter receptor and the peripheral olfactory system provides crucial information that may contribute toward novel therapy for anosmic and hyposmic patients who have compromised sense of smell, and to reduce stress associated with malodor, improving their quality of life.

The sense of small (olfaction) holds in it a major mystery: No scientist or perfumer can look at the structure of a novel molecule and predict its odor or molecular structure. This project aims to develop molecules that bind to individual olfactory receptors in the nose and thereby help solve the code by which odors result in odor perception. The proposed approach has broad implications and applications: It may pave the path towards the introduction of odor into everyday devices. The idea of odor-emitting televisions, computer game boxes, cell-phones, etc, has existed for some time. However, this goal remains unattained, mostly for "simple" technical reasons: Even if one successfully generates an odor-emitting device, what does one then do with the emitted odor? For example, a car-racing computer game may emit the smell of burning rubber tires, but how does one then evacuate the smell of burning rubber from the room? Moreover, given that odors linger, how can one rapidly switch from one odor to the next? The technology proposed here may solve these problems because it will entail particles that are odorless, yet take on a given odor as a function of rapidly-switchable externally applied fields. If successful, the proposed mechanism will drive a revolution of odor devices. Further, the "switchable chemical" approach will be extendable to other receptors in the brain, and can be applied towards asking basic questions concerning emotion, sensorimotor-coordination, memory and learning, as well as developing potential novel therapies for diseases associated with receptor signaling failure.

To develop a path towards solving the combinatorial code of olfaction, the Bachelet lab will design DNA strands called aptamers that assume a 3D structure that will specifically bind to a single type of olfactory receptor and induce signal transduction. These DNA-based "artificial odorants" will be tagged with a nanoparticle that changes its conformation in response to an external electromagnetic field. The product will be artificial odorants that are externally switchable in vivo. The Matsunami lab will use tissue culture cells expressing olfactory receptors to validate the function and selectivity of these switchable nano-robot odorants. The Sobel lab will then apply these artificial odorants to the human olfactory system, and measure perception and neural activity following switching the artificial odor on and off. This three-level approach will allow closure of the loop from receptor to perception, and potentially answer in this way what remains a fundamental question in olfaction.

This project was developed during a NSF Ideas Lab on "Cracking the Olfactory Code" and is jointly funded by the Chemistry of Life Processes program in the Chemistry Division, the Mathematical Biology program in the Division of Mathematical Sciences, the Physics of Living Systems program in the Physics Division, the Neural Systems Cluster in the Division of Integrative Organismal Systems, the Division of Biological Infrastructure, and the Division of Emerging Frontiers.

The mammalian sense of smell is arguably the most complex sensory system in the animal kingdom. Hundreds of olfactory receptors are deployed to detect a vast array of chemicals with exquisite sensitivity in complex environments. This collaborative project combines biochemistry, neurobiology, genomics, mathematics and new technologies to understand how the mammalian olfactory system detects, encodes and extracts meaning from chemical stimuli. The goals of this project are to: (1) elucidate fundamental neural mechanisms for how chemical sensation turns into the perception of a smell; (2) produce a vast array of scientific resources to olfactory scientists; (3) provide valuable information for broader audiences, including for molecular evolution, chemical ecology, and flavor and fragrance communities; (4) establish new technologies and mathematical frameworks to study biological systems; and (5) facilitate applied chemical sensing technologies for environmental monitoring, food safety, and homeland security. The project also offers training opportunities from the high school to the postdoctoral trainee level, and educational opportunities and outreach through partnerships with local science museums as well as science learning centers and their media outlets.

This project's efforts are organized around three aims that focus on how information about odor identity and odor valence (attractiveness/aversiveness) is encoded at the level of olfactory receptors (Aim 1); within the olfactory bulb, where odor information is first processed (Aim 2); and the cortical amygdala, where odor codes may integrate with other information streams (Aim 3). Completion of the project entails the development and use a broad array of innovative approaches that include mapping all human and mouse odorant receptors to the chemicals they bind, defining the innate valence of these chemicals using behavioral assays, mapping all odorant receptor projections to the olfactory bulb, functionally characterizing their neural representations in the olfactory bulb and cortical amygdala, and using novel mathematical approaches to understand the underlying structure of odor coding and olfactory neural circuits at the level of sensory neurons, olfactory bulb glomeruli, and amygdala. Progress towards each aim involves close collaborations between team members with diverse expertise, including molecular biology, behavioral neuroscience, in vivo functional imaging, and mathematical and theoretical analysis of complex datasets. The multidisciplinary strategy implemented here promises to lead to an integrated and comprehensive understanding of how mammals sense and make sense of their chemical environments.

The human sense of smell (olfaction) is central to many human activities and likely has a more complicated role in evolutionary pathways than previously suspected. Some examples of the important role olfaction plays in human behavior include detecting the fat content of food, directing food (and possibly mate) preference, and assessing health. Human olfaction is different from our closest living ape relatives, but it is not yet known if it is similar to or distinct from our extinct hominin relatives. The fundamental issue addressed by this project is whether or not genetic variation underlying odor detection receptors in the noses of modern humans, Neandertals, and Denisovans led to differences in how each species smelled the world. The investigators will use an innovative bioinformatics and laboratory approach to uncover rebuild key components of ancient noses. On a broad scale, the project promotes progress in the science of ancient genomes and evolutionary biology by assessing gene function in extinct populations. The data generated will contribute to human evolutionary genetics, biology, and anthropology. Application of these methods to other ancient genes has potential to transform our understanding of key evolutionary changes in humans, including genes implicated in human health and disease susceptibility. Finally, the project engages in public outreach to promote science education via a variety of national and international events, and includes funding to support training and professional development for under-represented individuals in STEM disciplines.

This EAGER project will investigate whether the olfactory repertoires of ancient hominins are distinct from or overlap with modern humans. Specifically, the investigators will determine if novel variants in the paleogenomes of Altai Neandertals and Denisovans resulted in distinct olfactory repertoires, using variant calling in the paleogenomes of these two hominins, reconstruction of 30 paleo-olfactory receptors via overlap extension polymerase chain reaction, and experimental validation of functional responses to odorants via a high throughput Dual-Glo Luciferase Assay System. The study will be one of the first to validate function of paleogenes and the first to do so on a large number of genes related to olfaction, an understudied area of human evolution. Identifying variation in olfactory repertoires within a comparative evolutionary framework may advance our understanding of hominin dietary evolution and evolutionary ecology. The investigators introduce a method that could be used by other researchers to validate function in other paleogenes that might shed light on modern human health problems, using paleospecies as a model (alongside others) for gene function.

DESCRIPTION (provided by applicant): The mammalian olfactory system has evolved to detect and discriminate amongst a myriad of volatile odor molecules as well as to respond to chemical cues from food, toxins, mates or predators to maximize fitness by utilizing a large family of odorant receptor (OR) proteins, members of G protein- coupled receptors. The mapping between ORs and corresponding odorants is, however, very complex: a given OR can be activated by more than one odor, just as one odor can activate an ensemble of ORs. The large number of receptors and odors, together with the multiple-to-multiple mapping relationships, make the systematic identification of odor-OR pairs a daunting task. Despite the success our lab and others have had in mapping odors to receptors by heterologous cell expression techniques, few studies have validated these in vivo. Here, we propose to develop a method to map ORs activated by odor stimulation in awake behaving animals, and use the method in combination with functional expression of ORs expressed in heterologous cells to identify a large set of odor-odorant combinations. This project will advance our understanding of peripheral odor coding by identifying the receptor repertoires of diverse odors, some of which elicit stereotypical behaviors. The resources generated by this study will be fundamental to many researchers in the olfaction field, who are interested in odor coding, behavior, and pre-receptor events. This new strategy aimed at comprehensively identifying a repertoire of ORs will be broadly applicable to the study of many other odorants and across diverse species.

Smell is a powerful sense that can trigger intense emotion, stereotyped behaviors and durable memories. The sense offers an extraordinary opportunity to connect atomic-level objects (odorant molecules and smell receptors in the nose) to neural responses. This project will predict which smell receptors in the nose are activated by a given odor. To accomplish this goal, the team of investigators will apply computational approaches to develop chemical structure-based receptor models and test these models using odor molecules interacting with olfactory receptors. The success of the project will enable the team to understand more precisely how the brain perceives the external environment. The results will also have widespread and diverse industrial applications, including rational design of new flavors and fragrances and development of new biosensors for detecting various chemicals. Furthermore, this project will make broader impacts in training and educating high school, undergraduate, and graduate students in various disciplines as well as in outreaching activities.

The complexity of the odor molecules, the large number of the smell receptors and combinatorial activation of the receptors make understanding odor coding an enormous challenge. This collaborative proposal represents the first of its kind that combines computational approaches with experimental measurements at both the receptor and the neuron level. Affinity calculations between odorants and the receptors, as well as the receptors' activation, will be obtained by nanosecond-scale simulations. Atomic-level simulations, initially assessed by experiments, will predict which odors would activate the receptors of interest. Comparisons between experimental findings and computational predictions will lead to a comprehensive computational model that converges with experimental data.

A companion project is being funded by the French National Research Agency (ANR).

Biogenesis of G protein-coupled receptors (GPCRs), the largest molecular class of pharmaceutical targets for cardiac, psychiatric, and cancer diseases is a complex and underexplored process. Many GPCRs require the presence of specific accessory proteins or co-receptors for their cell surface trafficking and/or functional expression. We previously identified RTP1 (Receptor Transporting Protein 1) and RTP2, both of which are single transmembrane proteins strongly and exclusively expressed in the peripheral olfactory organs. They greatly enhance trafficking of olfactory receptors (ORs) on the cell surface and induce functional expression in heterologous cells. In preliminary studies we investigated the roles played by the RTPs in vivo using the RTP1 and RTP2 double gene knockout mice (RTP1,2(-/-)), and uncovered an unexpected link between OR trafficking and OR gene choice mediated by the RTPs. Based on these and other preliminary studies, we will test three hypotheses. These are 1) RTP1 and RTP2 influence OR gene choice, 2) RTP mutant animals use diminished repertoire of functional ORs, and 3) specific residues of ORs regulate ER retention and cell surface trafficking. The successful completion of the project will deepen our understanding of OR biogenesis and function, and sheds light on the new link between OR protein trafficking and OR transcriptional regulation.

US-Japan Collaboration in Computational Neuroscience, Osaka, November 29-30, 2011

This award supports a workshop, led by Hiroaki Matsunami and Izumi Ohzawa, on US-Japan collaboration in computational neuroscience. The workshop builds on the interests of multiple NSF directorates and NIH institutes, and Japan's National Institute of Information and Communications Technology (NICT) and Center for Information and Neural Networks, in this rapidly developing area of research.

The workshop will explore the intellectual opportunities, broader impacts, and practical considerations needed for US-Japan collaboration to be successful. It will be attended by researchers and government representatives from the US and Japan. A report from the workshop will be made available at http://www.nsf.gov/crcns.

Copper (Cu) is an essential trace metal that is acquired from the diet and serves as a catalytic co-factor for a wide variety of enzymatic reactions that play critical roles in life. Cu deficiency leads to pathophysiological manifestations including impaired iron absorption, neutropenia, cognitive defects, peripheral neuropathy and hypertrophic cardiomyopathy. Understanding the mechanisms responsible for the accumulation of Cu in cells and tissues, the regulation of Cu accumulation, and the consequences due to dysregulated Cu acquisition are important to human health. Ctr1 is the only known Cu+ importer in mammals and while Ctr1 plays an essential role in dietary and peripheral Cu acquisition, embryonic development, cardiac function and normal growth, little is known about the mechanisms that regulate Ctr1 activity. Ctr1 exists both as a full-length protein and as a truncated form (tCtr1) lacking the extracellular Cu binding domain, which has reduced Cu uptake activity. We identified cathepsin as a protease that carries out the rate-limiting step in Ctr1 ecto-domain cleavage and demonstrated that this cleavage is stimulated by the Ctr2 integral membrane protein. Among patients in a large cardiac catheterization clinic cohort, we identified a single nucleotide polymorphism (SNP) in the human Ctr1 gene that occurs predominantly in African Americans, resulting in hyper-cleavage of the Ctr1 Cu-binding ecto-domain and decreased cellular Cu acquisition. Here we detail experiments to test the hypothesis that Ctr1 ecto-domain cleavage, through the cathepsin L/B proteases and Cu-responsive Ctr2 levels, is a critical regulatory mechanism for mammalian Cu acquisition. Our experiments will identify new components in mammalian Cu homeostasis, decipher a new mechanism for Cu- dependent proteolysis, generate a new animal model and validate a link between a defect in Ctr1 ecto-domain cleavage, Cu deficiency and hypertrophic cardiomyopathy in African Americans.