Timothy S. McClintock, Ph.D. - US grants

Recent experimental evidence suggests that the outer hair cells of the mammalian cochlea act as electromechanical amplifiers which increase hearing sensitivity one-hundred-fold. The long term goal of the proposed research is to confirm this hypothesis and to clarify our understanding of the underlying mechanisms. The specific aim of this proposal is to obtain experimental evidence in support of a detailed hypothesis describing the function of the cochlear amplifier. Our specific hypothesis is that the outer hair cell receptor current regulates a force-generation process which is located in the hair cell stereocilia. This force is coupled to the basilar and tectorial membranes and significantly increases the mechanical stimulus to the inner hair cells. We plan to stimulate the force-generation process through the injection of electrical current into scala media of the cochlea and to measure the resulting mechanical response as an otacoustic emission at the tympanic membrane. We will also use acoustic stimulation and measure the cochlear microphonic to study the role of the outer hair cell receptor current. We will study the effects of experimental manipulations which modify the amplification process. The effects of stimulation of the cochlear efferents on the force-generation process will also be studied. The results of the proposed experiments as well as previous experiments by ourselves and others will be interpreted with the aid of computational models. The computational models are physically based and include specific descriptions of the properties of the basilar and tectorial membranes as well as the hair cell transduction processes.

Functional domains and expression of olfactory receptors. The family of olfactory receptors determines the specificity of activation of olfactory receptor neurons, thereby defining the neural coding of every odor perception. This proposal describes studies of functional molecular domains of olfactory receptor proteins. The first objective is to determine the specificity of G-protein interactions of two or more olfactory receptors. These experiments will test hypotheses about the location of the domains which interact with G-proteins, their structures, the amino acids which determine coupling in these domains, the spatial relationships between the domains and between the important amino acids. Preliminary data from olfactory/beta2-adrenergic receptor chimeras predIct that olfactory receptors are capable of interacting with the G-proteins of heterologous cells and that olfactory receptors cause the production of cAMP. These functional expression experiments are performed in a novel assay system which uses optical methods (video imaging and microplate reader) to detect the second messenger-activated movement of pigment organelles in melanophore cell lines. This assay system is capable of efficiently screening thousands of odorants against dozens of olfactory receptor clones to determine odor-response spectra. These spectra are crucial to understanding the neural coding of odor stimuli. Preliminary data show that olfactory receptors are poorly expressed in the plasma membrane of melanophores. These findings may explain the lack of progress in functional expression studies using a variety of heterologous cell types. The second objective is therefore to investigate the molecular basis for the inability of olfactory receptors to be expressed in the plasma membrane of melanophores. The hypotheses to be tested are that olfactory receptors possess an internal sorting or folding signal that is misinterpreted by heterologous cells and that an olfactory-specific accessory protein is necessary for the proper expression of olfactory receptors. This proposal represents a systematic approach toward understanding the odorant and G-protein specificity of a large number of olfactory receptors. As such, this project will have direct implications for our understanding of anosmias and their impact upon the quality of human life.

In the highly heterogeneous central nervous system, the genes of greatest interest are those involved in the unique functions of each neuronal phenotype, and with their dysfunctions related to disease, aging, and injury. The objective of this project is to implement a generalizable stategy to identify these genes. The first aim is to develop methods to isolate phenotypically similar neurons and specifically identify cDNAs from mRNAs that are critical to phenotype and function. Transgenic mice expressing degradable green fluorescent protein (dGFP) from the per1 promoter, a clock-element gene, or from the fos promoter, a clock-driven gene, allow automated isolation of suprachiasmatic nucleus neurons expressing a circadian rhythm. Developing methods to use antisense RNA amplification, a linear mechanism of cDNA amplification from single neurons, and representational difference analysis, a high fidelity method of differential cDNA subtraction, on single phenotypically sorted neurons will allow both broad characterization of the mRNAs they express and specific identification of mRNAs whose expression changes during the circadian rhythm. Rather than assuming that changes in mRNA expression necessarily lead to protein changes, the second aim will reduce to practice a novel method for improved sensitivity in Western blots and use it to test rhythms in protein expression. The third aim seeks to develop a method to simultaneously measure multiple proteins from single neurons, using a high-throughput optical method that is predicted to detect antigens at levels sufficient for analysis of proteins in single neurons.

DESCRIPTION (adapted from applicant's abstract): The goal of this research is to describe molecular changes that occur during early and intermediate steps in the neurogenesis of olfactory receptor neurons of adult mice. The olfactory receptor neurons are one of the few populations of neurons that undergo continuous replacement in adult vertebrates. While a few examples of gene expression changes in the olfactory receptor neuron lineage are known, they represent only a minute fraction of the changes that must occur. Advances in recombinant DNA technology now make it possible to simultaneously identify large numbers of differentially expressed genes without a priori sequence information. Therefore, the investigator's aim is to use this technology to identify mRNAs that increase in olfactory epithelia during massive, synchronous neurogenesis of olfactory receptor neurons. This will include identifying mRNAs that are differentially abundant between early and intermediate stages in the process of neurogenesis. These experiments have the potential to: 1) identify a significant fraction of the genes involved in neurogenesis of olfactory receptor neurons; 2) identify signaling pathways that regulate neurogenesis; 3) expand the number of markers of cell types in the olfactory receptor neuron lineage; and 4) identify previously unknown cell types in the lineage. Within olfaction, the experiments have implications for embryonic development of the olfactory epithelium, for olfactory carcinomas, for regeneration, and aging processes in the olfactory epithelium. The experiments also have implications for neurogenesis in adult mammals in general, including using this knowledge to develop interventions in neurodegenerative disorders and traumatic injury to the nervous system.

[unreadable] DESCRIPTION (provided by applicant): The sensory neurons of the mammalian olfactory epithelium can be regenerated even if they are completely eliminated. This capacity, which lasts throughout the life of the animal, is of significant interest for both basic and applied neuroscience research. Its mechanisms are presumed to be relevant to the search for regeneration-promoting therapies for neurodegenerative disorders and trauma of the nervous system. To extend our understanding of olfactory regeneration, we have identified 1,260 transcripts whose abundance within the olfactory epithelium changes after removal of the olfactory bulbs, a manipulation that causes the selective death and subsequent regeneration of the olfactory sensory neurons. Of these transcripts, 303 increase contemporaneously with the proliferation of sensory neuron progenitors. The functions of the proteins encoded by these 303 transcripts predict several underlying processes, including transcriptional activation of cell proliferation and differentiation, up-regulation of the cell cycle, axon outgrowth, and cell signaling. Only a handful of these proteins have previously been linked to olfactory regeneration. In this application, we propose to focus on a subset of gene products that are likely to be critical for proliferation and differentiation of the olfactory sensory neurons. The first specific aim is to define the capacity for each transcript to be involved in olfactory regeneration by determining which cell types express them, including spatial and temporal overlap with markers of known progenitor cell types. The second and third aims focus further on a small subset of genes for which targeted deletions are available. These aims test whether the absence of the gene alters the development or regeneration of the olfactory epithelium, leading to changes at the behavioral, cellular, or molecular level. The proposed experiments will definitively test whether several proteins are dispensable for olfactory regeneration and will define the potential roles of a couple dozen additional proteins. [unreadable] [unreadable]

The sensory neurons of the olfactory epithelium can be regenerated even if they are completely eliminated. This capacity lasts throughout the life of the animal and is of significant interest for both basic and applied neuroscience research. Its mechanisms are presumed to be relevant to the search for regeneration- promoting therapies for neurodegenerative disorders and trauma of the nervous system. Its mechanisms are also clearly relevant to neural development. To extend our understanding of olfactory regeneration, we have identified 1,205 mRNAs whose abundance within the olfactory epithelium changes after removal of the olfactory bulbs, a manipulation that causes the selective death and subsequent regeneration of the olfactory sensory neurons. Of these mRNAs, 303 increase contemporaneously with the proliferation of sensory neuron progenitors. The functions of the proteins encoded by these 303 mRNAs predict several underlying processes, including transcriptional activation of cell proliferation and differentiation, up-regulation of the cell cycle, axon outgrowth, and cell signaling. Only a handful of these proteins have previously been linked to olfactory regeneration or development. In this application, we propose to focus on a subset of gene products that are likely to be critical for proliferation and differentiation of the olfactory sensory neurons. The first specific aim is to define the capacity for each gene product to be involved in olfactory regeneration by determining which cell types express them, including spatial and temporal overlap with markers of known progenitor cell types. The second and third aims focus further on a subset of genes for which targeted deletions are available. These aims test whether the absence of the gene alters the development or regeneration of the olfactory epithelium, leading to changes at the cellular or molecular level. The proposed experiments will definitively test whether several proteins are dispensable for olfactory development and regeneration, and will define the potential roles of a couple dozen additional proteins.

Description (provided by applicant): The support cells of the olfactory epithelium have received little direct attention, yet several lines of evidence indicate that they are critical for maintaining or regulating the olfactory sensory nerve cells, the ongoing replacement of olfactory sensory nerve cells, and the local environment that allows odor detection. A detailed molecular understanding of the two major types of support cells, sustentacular cells and Bowman's gland cells, is lacking. Fortuitously, we have developed a mouse strain that will allow us to extract two dissociated cell samples: One highly enriched for sustentacular cells, the other for Bowman's gland acinar cells. Aim 1 will therefore investigate whether two support cell transcriptomes show overrepresentation of biological processes hypothesized to be active in these support cells: Clearance of foreign chemicals, cell adhesion, clearance of dying cells, secretion, and certain cell signaling pathways. The outcome of this aim will also include a database of the probability of expression in sustentacular cells and Bowman's gland acinar cells for more than 10,000 genes. Aim 2 delves more deeply into a mechanism we hypothesize controls phagocytosis by sustentacular cells, one of the few documented functions of these cells. We hypothesize that signaling through the receptor tyrosine kinase Tyro3 regulates clearance of dying nerve cells by sustentacular cells. Overall, this project is another step toward a complete understanding of gene expression patterns in the olfactory epithelium and will identify most functions present in the little-studied support cells of this tissue. In addition, these experiments are relevant to age-dependent loss of olfactory function because deficits in Tyro3 receptor family signaling in the support cells of several other tissues are known to lead to age-related disorders, such as retinal degeneration. PUBLIC HEALTH RELEVANCE: The olfactory system has evolved to detect and discriminate many thousands of distinct volatile chemicals, in part by generating a favorable microenvironment and retaining the ability to replace damaged olfactory nerve cells even as aging occurs. Investigating how the support cells of the epithelium contribute to odor detection and nerve cell replacement is bound to improve our understanding of how cellular interactions contribute to the ability of the olfactory system to function throughout an animal's life span. This project investigates the molecular capabilities of the support cells of the epithelium and a hypothesized mechanism by which support cells clear dying nerve cells to make way for new nerve cells.

DESCRIPTION (provided by applicant): The application proposes to allow an investigator experienced in the molecular biology of the olfactory epithelium to cross the gap from his expertise in microarray-based genomics to more computationally challenging next generation sequencing approaches. The plan includes: (1) training in R programming to allow the investigator take advantage of flexible analysis methods for ChIP-seq and RNA-seq techniques, (2) mentored pilot experiments in both of these approaches, and (3) mentored analyses of the data. The pilot studies take advantage of a unique resource that allows purification of olfactory neurons expressing subsets of odorant receptors to work toward testing two fundamental hypotheses about odorant receptors. (1) Active chromatin modifications (which can only be detected by enrichment for neurons expressing an odorant receptor) are critical for the tightly regulated expression of odorant receptors. Active chromatin modifications will be measured by ChIP-seq. The exquisitely specific control of odorant receptor expression is perhaps the greatest remaining mystery about the function of olfactory sensory neurons. (2) Odorants activate sets of odorant receptors that may overlap but must have at least one distinct member if the odorants can be discriminated. Enrichment for olfactory sensory neurons marked by expression of GFP from an activity-dependent gene locus will allow RNA-seq methods to identify sets of odorant receptors activated by specific odorants. Understanding the fundamental question of how hundreds of odorant receptors allow the detection and discrimination of thousands of odorant chemicals should help explain some causes of hyposmia and anosmia; and should allow the future development of odorant agonists and antagonists that could be used to improve the quality of human life.

DESCRIPTION (provided by applicant): This project investigates the actions of two homeodomain transcription factors that help control expression of nearly all of the more than 1,000 mouse odorant receptor genes, expression so finely regulated that each olfactory nerve cell expresses just one allele of one odorant receptor gene. The overarching hypothesis for the project proposes that homeodomain transcription factor stimulation of odorant receptor gene transcription triggers local positive feedback acting through active chromatin modifications while simultaneously enhancing global negative feedback acting through repressive chromatin modifications, competing events capable of explaining the singularity of odorant receptor expression. This project tests whether the transcription factors act directly at promoters and enhancers of odorant receptor genes, and whether these events are associated with selected active chromatin modifications at odorant receptor genes.

ABSTRACT The objective of this conference grant request is to provide support for certain aspects of the 2nd annual meeting of the International Society of Neurogastronomy (ISN). ISN is a new society whose composition of scientists, clinicians, nutritionists, food service professionals and chefs naturally predisposes it to focus on translational issues fundamental to how the chemical senses impact human health. The first two specific objectives are to provide a venue and opportunity that brings these groups together and facilitates the synergies that arise when distinctly different fields interact productively. The third specific objective is to attract and stimulate the young scientists that are the future of the field of neurogastronomy. The fourth specific objective is to ensure that this new field embraces a culture of diversity, weaving this principle into its early development by recruiting persons of diverse backgrounds to the conference.

? DESCRIPTION (provided by applicant) This project uses a new in vivo assay that allows the identification of the odorant receptors activated in live mice by any odor, making it possible to identify in one experiment many receptors activated by the odor. This accelerates the identification of receptors sensitive to any odor and when combined with in vitro assays for the function of individual odorant receptors, allows testing of hypotheses about the sets of receptors activated by odorants and odor mixtures. The first aim targets a specific region of the broad universe of odorants, testing whether the overlap in sets of receptors responsive to these odorants depends on their structural similarity. The second aim tests hypotheses about the possible interactions between odorants at odorant receptors. It begins with pairs of odorants that are known or suspected of interacting, mostly by the ability of one odorant to block activation of one or more receptors by another odorant. It then moves on to testing more complex mixtures, seeking to identify interactive effects between odorants at receptors - effects that are fundamental to the characteristic sensation of the odor and the discrimination of one odor from another. In addition to improving our understanding of the mechanisms of odor perception, these experiments provide knowledge expected to facilitate the development of more sophisticated ways of manipulating odor environments to benefit the quality of human life.

ABSTRACT The objective of this conference grant request is to provide support for certain aspects of the annual meeting of the International Society of Neurogastronomy (ISN) for the next five years. ISN is a new society whose composition of scientists, clinicians, nutritionists, food service professionals and chefs naturally predisposes it to focus on translational issues fundamental to how the chemical senses impact human health. The first two specific objectives are to provide a venue and opportunity that brings these groups together and facilitates the synergies that arise when distinctly different fields interact productively. The third specific objective is to attract and stimulate the young scientists that are the future of the field of neurogastronomy. The fourth specific objective is to ensure that this new field embraces a culture of diversity, weaving this principle into its early development by recruiting persons of diverse backgrounds to the conference. !

PROJECT SUMMARY/ABSTRACT The parent award for this administrative supplement request investigates the patterns of odorant receptor responses evoked by odors and the cell and molecular biology of the olfactory sensory neurons that express these odorant receptors. Deficits in olfactory performance are an early symptom for many patients suffering from Alzheimer?s Disease (AD) and AD patients typically show neuropathology in brain regions that process olfactory information, including the loss of olfactory sensory neurons. This susceptibility of olfactory sensory neurons to AD-related neurodegeneration is a fundamental reason they are advantageous models for investigating the onset and progression of AD-related neurodegeneration. Another fundamental reason is our highly detailed understanding of the biology of these neurons, such as knowing the identity of all genes they express. Other reasons include their accessibility for experimental manipulation, their homogeneity and large population size (>5 million per mouse), and the numerous genetically modified mouse strains that have been made to study these neurons. Investigating the onset of AD is critical given that therapies targeting late stages of the disease have been unsuccessful. Olfactory sensory neurons are particularly well-suited for determining the onset and order of progression of events responsible for AD-related neurodegeneration. We propose to take advantage of our ongoing study of olfactory sensory neuron function to carefully and deeply investigate phenotypic changes in olfactory sensory neurons in two types of mouse models of AD. We expect to identify major events in the early stages of AD-related neurodegeneration and thereby demonstrate that olfactory sensory neurons are a powerful model for study of the onset of AD.

ABSTRACT The objective of this conference grant request is to provide support for certain aspects of the annual meeting of the International Society of Neurogastronomy (ISN) for the next five years. ISN is a relatively new society whose composition of scientists, clinicians, nutritionists, agricultural experts, food service professionals, and chefs naturally predisposes it to focus on translational issues fundamental to how the chemical senses impact human health. The first two specific objectives are to provide a venue and opportunity that brings these groups together and facilitates the synergies that arise when distinctly different fields interact productively. The third specific objective is to attract and stimulate the young scientists that are the future of the field of neurogastronomy. The fourth specific objective is to do outreach that serves health and culinary professionals by informing them about the impact that smell and taste has upon diet, nutrition, and the growing health problems of obesity-related diseases. The fifth specific objective is to ensure that this new field embraces a culture of diversity, weaving this principle into its early development by recruiting persons of diverse backgrounds to the conference.