1998 — 1999 |
Feinstein, Paul |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Determinants of Connectivity in the Olfactory System
Olfactory receptors are encoded by a family of 1,000 or more genes in mice. An olfactory sensory neuron expresses most likely only one of these genes. Neurons expressing a given receptor are scattered within one of four zones in the olfactory epithelium; the biological significance of the zonal pattern remains unknown. Axons of neurons expressing a given olfactory receptor project to two out of 1,800 glomeruli in the olfactory bulb, posing a formidable wiring problem. Previous studies by the sponsor have implicated the olfactory receptor itself in connectivity, but it cannot be the only determinant in the guidance process. In preliminary studies the applicant has focused on the M71 and M72 genes, which encode two highly related receptors that are expressed in the same epithelial zone. The M71 and M72 glomeruli were shown to be distinct. Here, a continuation of these experiments is proposed that seeks to define and describe two determinants of connectivity in the olfactory system: the epithelial zone in which the neurons are located, and the olfactory receptor itself. Gene targeting technology will be used to engineer mice such that they express a variety of knock-in mutations, mostly in the M71 gene. The experiments differ from previous studies by the sponsor in that genetic manipulation is carried out with olfactory receptor genes that are expressed in the same zone.
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0.943 |
2005 — 2007 |
Feinstein, Paul |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Development of Olfactory Glomeruli
[unreadable] DESCRIPTION (provided by applicant): The coding of olfactory information begins with an animal's ability to detect and discriminate odorants. In the mouse olfactory system this is accomplished by a family ofapproximately 1000 functional odorant receptor genes each of which is expressed in thousands of neurons. A mature olfactory neuron expresses only 1 odorant receptor gene from only 1 allele. Odorant receptors (OR) are critical for the convergence of thousands of identical axons into 2 symmetrically located glomeruli on an olfactory bulb thereby creating approximately 2000 glomeruli. The marriage of mouse genetics and neural development has allowed for molecular dissection of these processes. This project continues with the analysis of the mouse olfactory system. In Aim 1, the temporal development will be determined for 2 highly related ORs that correspond to spatially related positions on the surface of the olfactory bulb. This analysis will determine if the onset of glomerular formation correlates with its final position on the olfactory bulb. A parallel study will determine if altering the number of axons for a given axonal population affects its position of convergence. In Aim 2, polymorphic OR proteins will probe if domains in the OR impart specificity to axons that converge into glomeruli. These experiments will provide a foundation for how olfactory glomeruli develop. Results generated will elaborate whether there are intrinsic positions on the olfactory bulb that are laid down by gradients and if there is an under appreciated plasticity in the olfactory system. The process by which olfactory sensory neurons are constantly re-establishing codes of connectivity to the olfactory bulb, may give insights into axonal pathfinding in other regions of the brain. [unreadable] [unreadable]
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1 |
2010 — 2011 |
Feinstein, Paul |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
High Throughput in Vivo Functional Analysis of Human Odorant Receptors.
DESCRIPTION (provided by applicant): The human olfactory system is thought to detect thousands of odorants, allowing for proper food consumption and aversions. The ability to identify odors by humans is dependent on odorant receptors within the olfactory neurons that line the nasal epithelium. Odorant receptors (ORs) are clonally expressed in mature olfactory neurons (one OR allele per neuron). Thus studying odorant responses in identifiable individual neurons is equivalent to studying individual ORs. The human olfactory genome consists of ~900 OR genes;these genes belong to the seven-transmembrane receptor superfamily. However, only ~40% encode a full-length open reading frame (ORF). Thus, ~355 OR genes are available for odor identification in the main olfactory system. This subgroup of ORs breaks down into three OR Classes: Class I: ~50 genes;Class II: ~300 genes;and the TAARs: ~5 genes. With the emergence of SNP analysis, the number of variant, human ORs with full-length ORFs continues to grow. The entire set of Class I and II OR genes have been known for a decade, yet their ability to function in vivo has remained elusive. Three things are severely lacking in order to understand the functionality of all ~355 OR genes plus their variants: 1st) knowledge of which ORs are expressed in human olfactory neurons 2nd) evidence that the ORFs can couple to the olfactory G- protein (Golf) in an in vivo setting and 3rd) evidence that the ORFs can allow for glomerular formation in an in vivo setting. Failure for some human OR ORFs to drive glomerular formation or couple to Golf would classify these genes as "pseudogenes". Thus ORs having a full-length ORF does not constitute a valid enough reason for studying their odorant binding properties using in vitro technologies. There needs to be a method for quickly identifying which human OR ORFs can function in vivo and simultaneously make use of this in vivo system for the identification of odorant-OR activity correlates. In Aim 1 section A, a high-throughput gene-targeting strategy will be developed such that human OR ORFs can be rapidly "swapped" in place of a mouse OR ORF and be tested for functionality in vivo. Initial experiments will determine the efficacy of this approach at two mouse OR loci. The intent of this sub aim would be to generate a library of Embryonic Stem cells that each contain a unique human OR ORF. Initially, several isoforms for one human OR will be tested. In Aim 1 section B, a high-throughput transgenic approach will be developed whereby 70% of all olfactory neurons (millions) in the mouse olfactory epithelium will clonally express a given human OR. Again, several isoforms for one human OR will be tested. This project is aimed at complementing the gene- targeting strategy and providing a large number of neurons for possible robotic approaches in odorant identification. In short, the sole aim of this proposal is to move forward our understanding of which human receptors are functional for human perception of odors. PUBLIC HEALTH RELEVANCE: The ability for humans to detect odors (odorants) remains an important aspect of human health and welfare. The sense of smell is critically dependent on hundreds of different human odorant receptors normally expressed in olfactory neurons. However, very few odorant/odorant receptor matches have been made. This lack of knowledge is a sole reflection of the inability to express and characterize human odorant receptor proteins in non-olfactory cells. Thus, the functional analysis of human odorant receptors expressed within olfactory neurons should significantly help bridge this divide.
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1 |
2010 — 2014 |
Feinstein, Paul |
SC1Activity Code Description: Individual investigator-initiated research projects aimed at developing researchers at minority-serving institutions (MSIs) to a stage where they can transition successfully to other s extramural support (R01 or equivalent). |
Regulation of Odorant Receptor Gene Expression
DESCRIPTION (provided by applicant): The olfactory system is able to recognize thousands of odorants thus maintaining food consumption in humans. The ability to identify odors by humans is dependent on odorant receptors within olfactory neurons that line the nasal epithelium. This epithelium is under continuous cell division by a basal stem cell population that produces immature olfactory neurons, which then become mature olfactory neurons. Odorant receptors (ORs) are clonally expressed in immature olfactory neurons. The mammalian genome consists of ~1500 OR genes;these genes belong to the seven-transmembrane receptor superfamily. However, each allele of each gene is treated as a separate entity leading to ~3000 OR alleles to be expressed in a clonal (singular) manner by olfactory neurons. The ability for this singular OR allele expression is a multi-step manner. A critical step is the production of a functional OR protein. Olfactory neurons are found in domains within the epithelium such that a given olfactory neuron chooses (in general) between ~100 OR genes (~200 alleles). The selection process occurs in two-steps: one of ~200 alleles is chosen for expression (1st choice) within an immature neuron, followed by OR protein production, which is tested for functionality. Failure to produce a seven-transmembrane protein allows for the singular choice mechanism to choose another OR allele in that same immature olfactory neuron (2nd choice). If the chosen seven-transmembrane OR is unable to couple with the signal transduction machinery, then that olfactory neuron will fail to mature and die. The critical step in the expression of an OR gene is the initial choice of one OR allele within an immature neuron (1st choice). To date, it is completely unknown how this "singular choice" process occurs at a molecular level. Analysis of very small OR promoter regions (~300bp) have lead to the identification of two transcription factors that may play a role: an O/E-1 type binding protein and a homeodomain type biding protein. The identification of more candidate DNA binding proteins and their interactions is necessary to understand the "singular choice" process. This project fully exploits the small regulatory regions used to produce OR singular choice in immature olfactory neurons. In Aim 1, the most minimal OR control region will be defined in order to identify other candidate transcription factors. There are at least 10-15 different olfactory neuronal cell types each fated to express one of ~200 OR alleles. In Aim 2, two minimal OR promoters normally expressed in different olfactory cell types will be used to identify additional transcription factors. This will allow the understanding of how specific patterns within the olfactory epithelium are obtained. In Aim 3, a candidate homeodomain protein will be deleted within immature and/or mature neurons in order to determine its role on OR gene expression. In Aim 4, I will make every attempt to promote this work into publications and greater funding. PUBLIC HEALTH RELEVANCE: Gene expression is necessary for cells to function properly in the human body. The sense of smell is critically dependent on olfactory neurons that translate the chemical world to the brain. Olfactory neurons govern this odor recognition process through careful expression of odorant receptors. Loss of olfactory function leads to poor feeding in geriatric populations as well as higher incidence of food poisoning. Studying odorant receptor gene expression will give insights into how the olfactory system is put together and maintained throughout adulthood.
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1 |
2018 — 2019 |
Feinstein, Paul |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Development of Genetically-Encoded Reporters For Olfactory Cilia Signaling
Abstract We have recently developed a genetic, customizable platform, called MouSensor, that can be used to express -for the first time- any odorant receptor protein (OR), including human ORs, in a large population of mouse olfactory sensory neurons, providing a breakthrough technology with a wide array of translational applications (D'Hulst et al., 2016). ORs expressed through this platform technology are fully functional and retain the one-receptor-per-neuron expression pattern. Moreover, behavioral tests show that our transgenic mice have an increased sensitivity for the specific odor that activates the cloned receptor. We ultimately envision transforming our mouse OR bioreactors into a bio-nose-on-a-chip by combining extracted MouSensor neurons with chip technology. The first technical hurdle we have to overcome in achieving this goal is to develop a reliable and quantitative objective analysis of OR activation ex vivo. In the olfactory system, when an odor activates an OR, a G protein cascade gets activated whereby cAMP levels rise dramatically leading to calcium entry through cyclic-nucleotide gated channels. Using genetically-encoded reporters for Calcium and/or cAMP is a feasible solution to report odor-evoked signaling. This entire cascade initiates in the cilia appendages attached to the dendritic knob; olfactory cilia can be readily detached from the neuron providing an ex vivo system to assess OR activation. The research proposed here allows us to develop an optical, objective read-out upon specific OR activation, by targeting genetically-encoded Calcium or cAMP reporters to olfactory cilia. Importantly, cilia have been shown to retain their activity after freezing, dramatically increasing the shelf-life of the assay. Here, we propose to target two ultrasensitive fluorescence- associated reporter proteins to primary cilia and provide a breakthrough technology to monitor Ca2+ and/or cAMP signaling in olfactory cilia by generating two different reporter mice. We will use CRISPR/cas9 genome editing to create fusion proteins to a cilia trafficking protein that we already know targets to cilia when fused to the Green Fluorescent Protein (GFP). Combining either of the new reporter lines with our established MouSensor technology will provide a streamlined system for high-throughput odor profiling and may lead to development of novel reporters for neuronal cilia signaling in general.
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1 |