James E. Schwob - US grants

[unreadable] DESCRIPTION (provided by applicant): The fundamental event during the differentiation of a peripheral olfactory neuron is the selective expression of one allele of one olfactory receptor (OR) gene from among the 1296 members of the superfamily of OR genes. The choice of OR will determine the stimulus sensitivity of the neuron and will determine where that neuron should send its axon, although the means by which the OR directs axon targeting is still poorly understood. This grant proposes to answer 3 fundamental questions about OR choice. 1) Precisely how does OR choice reflect spatial location in the olfactory epithelium? Preliminary evidence suggests that the conventional 4-zone model relating OR expression to location is insufficient. We will take advantage of the explosion of genomic information to test specific hypotheses relating OR family membership and chromosomal location to the pattern of expression. 2) Where does the memory of spatial position reside that allows reconstitution of OR expression after injury? We will transplant progenitor cells into foreign parts of the epithelium to see whether the progenitor cells encode a memory for place or whether cues that derive from the local environment direct expression. 3) How does OR choice govern axonal connectivity during the course of epithelial reconstitution and neuronal regeneration? Data gathered during the prior period of support suggests that recapitulation of the precise one OR-one glomerulus organization occurs during the recovery from extensive peripheral lesion only when a substantial population of pre-existing axons are spared. We will take advantage of strains of mutant mice in which expression of an OR is coincident with expression of a marker protein to extend our analysis of the consequences of mild, moderate, and severe lesions of the periphery. The data obtained will allow us to determine whether fasciculation of growing axons with spared like-axons occurs when the projection is restored to its pre-lesion precision, and fails when it is not. We will use both confocal microscopy and immuno-EM analysis to test our hypothesis. Successful completion of the aims has implications for our understanding and treatment of human olfactory disease. Attempts to foster recovery in the clinical population require an understanding in depth of the inherent limits and capacities for regeneration. [unreadable] [unreadable]

The peripheral olfactory system has a remarkable capacity for repair after injury, and the maintenance of that capacity depends on the persistence of neurocompetent stem cells. If the stem cells are destroyed, the injured tissue undergoes respiratory metaplasia, which is a major cause of sensory dysfunction in humans. Despite the importance attached to understanding the regulation of olfactory stem cells, there is much that is not yet known, starting with their identity. Work during the previous period of support demonstrated that among the population of globose basal cells (GBCs) are cells that have many of the characteristics of stem cells, including the potency to differentiate into all the constituent cell types and the existence of slowly cycling, label-retaining cells (a hallmark of stem cells in other tissues). The 3 Specific Aims proposed in this application will provide a more in depth understanding of olfactory stem cells and how they are regulated. Aim 1 will use a transplantation/colony forming unit assay to determine the differentiate and generative capability of different marker-defined subsets of GBCs, including label-retaining ones. Aim 2 will test whether expression of members of the bHLH transcription factor family signify (and might drive) irreversible commitment to a single lineage. Aim 3 will test whether the Notch-Hes signal transduction pathway controls the choice point between neurons vs. non-neuronal cells that is made by the multipotent GBCs as they differentiate. Successful completion of the Aims will advance our understanding of olfactory stem cells and hasten their potential use as a therapeutic modality.

A better understanding of the pathophysiological mechanisms underlying human olfactory dysfunction is obviously important for improving the clinical management of dysosmic patients. To that end we have been evaluating the histopathology to the human olfactory mucosa, and have been using that information to construct animal models of olfactory disease. This project continues our efforts and has the following Specific Aims; (1) To increase the available information regarding the histopathology of the olfactory mucosa in a variety of clinical settings, paying particular attention to (a) preservation of the neuronal population, (b) neuronal maturation, (c) aberrant axonal growth, (d) proliferation rate, (e) cell death. We will be focussing specifically on the mechanisms of epithelial regression (which we term quiescence), the type of neuronal damage in post- URI patients, and the pathology associated with phantosmia. (2) To identify the mechanism underlying epithelial regression in an animal model of human quiescence, namely mice made transgenic for an OMP-SV40 T antigen construct. During regression, we will analyze basal cell proliferation, neuronal lifespan and neuronal differentiation in order to achieve that aim. In parallel with the anatomical analyses, we will monitor the functional consequence os epithelial regression by assessing both behavioral performance on a 5 odorant identification task and the patterns of inherent mucosal activity as a function of age. (3) To assess whether IGF-1 will serve as a survival factors and extend the lifespan of olfactory neurons born in the absence of the olfactory bulb, on which the neurons are trophically dependent. At least in part, several forms of dysosmia are due to a lack of axonal connection between the epithelium and bulb and the accelerated turnover of neurons that occurs as a consequence. IGF-I- containing pellets will be implanted into the ablation cavity after bulbectomy and neuronal life-span will be determined by following the cohort of neurons labeled at birth via the incorporation of thymidine and by following the set of neurons labeled after axonal growth into the ablation cavity via the retrograde transport of fluorescent beads. (4) To assess whether transplanted basal cells will incorporate into the epithelium of a lesioned host and generate neurons, and whether those neurons will survive, differentiate and connect with the olfactory bulb. At least two forms of olfactory pathology --the permanent replacement of olfactory by respiratory epithelium and the regression of the olfactory epithelium -- are probably due to destruction or exhaustion, respectively, of the epithelium's neurogenic potential. Transplanted cells will express beta-galactosidase as a heritable marker, which will allow us to recognize them ad their progeny in the host tissue; heritability ill be conveyed by germline incorporation of the transgene or by infection of basal cells with a replication-incompetent retrovirus. Successful completion of these aims will provide substantial insight into the pathophysiology of human olfactory disease and open avenues for potential therapeutic intervention.

(Revised Abstract) DESCRIPTION (provided by applicant): The maintenance of olfactory function in humans as in other animals depends on the persistence of neurogenesis in the olfactory epithelium throughout life. If the progenitor population is destroyed during the course of an injury to the olfactory epithelium, the tissue undergoes metaplasia and reconstitutes as respiratory epithelium instead. We have shown that the globose basal cells (GBCs), among which are both broadly potent and neuronal-committed progenitors, can be selectively isolated by FACS and will engraft in the epithelium of a new host after transplantation. Accordingly, transplantation may be a viable strategy for restoring the epithelium's capacity for neurogenesis. Progress in implementing that strategy depends on the availability of a sufficiently large population of progenitors. To that end we are proposing experiments to test whether the pool of progenitors can be expanded in vitro and still engraft and differentiate appropriately in vivo. Two Specific Aims will be pursued. First, GBCs will be selectively isolated from normal mice that ubiquitously express GFP and others that have been lesioned with methyl bromide gas shortly before harvest. In the later case, the effect of the lesion is activate olfactory stem cells and to bias the population toward multipotency. The GBCs will be cultured in defined media with a melange of growth factors that have been shown to drive proliferation and/or neurogenesis in heterogeneous explant or dissociated cultures. Second, GBCs will be conditionally immortalized by transfection with a retroviral vector that encodes a temperature sensitive version of the large T antigen oncogene from SV40. The differentiation of the resulting clonal cell lines will be assayed after switching to the non-permissive temperature. Both the expanded primary isolates and cell lines that differentiate well after inactivation of the oncogene will be transplanted in order to define their capacity for differentiation in vivo (including a determination of whether donor-derived neurons express odorant receptors). Successful completion of the Aims will; 1) clarify the nature of growth factor control on olfactory neurogenesis; 2) determine whether cellular replacement is a viable strategy for restoring neurogenesis; 3) generate a set of protocols and cellular reagents that are useful for further studies of progenitor cell capacity and neuronal differentiation.

neural growth associated protein; protein structure function; developmental neurobiology; synaptogenesis; growth cones; recombinant proteins; enzyme activity; cellular polarity; actins; protein kinase C; nucleic acid sequence; intracellular transport; cytoskeleton; homozygote; gene mutation; genetic recombination; complementary DNA; phosphorylation; posttranslational modifications; calmodulin; phosphoproteins; southern blotting; tissue /cell culture; transfection; polymerase chain reaction;

DESCRIPTION (provided by applicant): The M.D. /PhD. program at Tufts University School of Medicine and the Sackler School of Graduate Biomedical Sciences is designed to train the physician scientists of the future, and provide an outstanding cadre of investigators well-versed in both the practice of medicine and the conduct of basic biomedical research. Our goal is to bridge the communication and cultural divide that too often separates physicians and scientists and educate individuals who will be firmly rooted in both cultures and achieve highly productive careers that meld these two arenas. Medical training interfaces with eight basic science graduate programs in Biochemistry, Cellular &Molecular Physiology, Cell, Molecular &Developmental Biology, Genetics, Immunology, Molecular Microbiology, Neuroscience, and Pharmacology &Experimental Therapeutics Programs that have a combined faculty of 135. The Tufts MSTP has been funded since 1991, but has undergone a major transformation in the past two years with the appointment of a new director and a new leadership team. A completely redesigned administrative structure, new admissions and advising processes, and major changes in the curriculum have been implemented. Our new curriculum effectively integrates research and clinical training. Research rotations prior to beginning medical school and research selective during the first two years of medical school allow students to make informed decisions about research mentors in a timely fashion. A new Clinical Connections program that provides an array of patient experiences during the years focused on research training has been put in place. A new course "Clinical Implications of Basic Research" that brings together M.D. /PhD. trainees in all phases of their training to discuss the ways bench research is being translated in new clinical practices has also been added. A workshop that provides orientation and hands-on experiences for students transitioning from research to clinical education will be given for the first time in the spring of 2006. A Retreat, seminars, and a career-oriented dinner program are also built into the training program. Our program has graduated 40 trainees since its inception and 32 are currently enrolled. Based on the changes that have occurred, we are requesting funding for 16 positions in this application.

[unreadable] DESCRIPTION (provided by applicant): The capacity of the olfactory epithelium (OE) to recover both anatomically and functionally after injury to near-normal, a capacity that extends throughout life, is unique in the nervous system. However, we know next to nothing about the molecular phenotype of the olfactory stems nor their downstream progenitors, an ignorance that severely hampers our ability to isolate them, understand their regulation, or use them in a controllable manner. We are proposing exploratory and developmental studies designed to address that vast gap in our knowledge. We will isolate three specific types of globose basal cells (GBCs), among whose functionally heterogeneous population, a broadly pluripotent, stem or stem-like cell is found: multipotent GBCs, sustentacular cell- forming GBCs, and transit-amplifying GBCs. The different kinds of GBCs will be defined by the timing of their re-emergence after methyl bromide lesion of the mouse OE and by their expression of one among a group of basic Helix-Loop-Helix (bHLH) transcription factors that will include Hes1 and Mash1. The bHLH TF-expressing cells will be isolated by FACS on the basis of markers on their surface that define them as GBCs and the expression of GFP in parallel with each individual TF (from either a transgen or the endogenous gene locus). For each TF-defined population we will carry out AffyMetrix microarray analysis designed to profile its pattern of gene expression. In addition, mitotically quiescent and BrdU label-retaining GBCs in the normal OE are a potential stem cell population. We will isolate them by FACS as well and subject them to gene profiling. As a consequence of these studies, we will have a comprehensive understanding of resting and activated olfactory stem cells and their downstream progenitors and will be poised to begin hypothesis-driven assessments of functional regulatory pathways. The stem cells of the OE are both easily accessible in adult humans and have a natural propensity to make neurons. Furthermore, the embryonic cells of the olfactory placode, which the adult stem cells resemble in both molecular and functional terms, give rise to the specialized glia of the olfactory nerve, which may promote functional recovery after spinal cord injury. The information gathered here may allow us eventually to use these adult stem cells in treating nervous system damage and degenerative disease. [unreadable] [unreadable] [unreadable]

Description (provided by applicant): Improvements in diagnosis and treatment of olfactory disease will require first, a more comprehensive analysis of the anatomical status of the olfactory periphery (the 1 cm2 areas on the nasal septum and the lateral nasal wall immediately inferior to the cribriform plate) with respect to the preservation of the olfactory epithelium (OE) and, second, a means of determining whether the OE's inherent capacity to reconstitute itself remains intact. We propose three specific aims designed to address our shortcomings with respect to the pathological bases of peripheral olfactory dysfunction. Aim 1: We observe a better than expected preservation of OE in the olfactory area of autopsy specimens, but immature neurons are abnormally prominent. We will test the hypothesis that areas in which immature neurons predominate are disconnected from the OB by using immunohistochemical analysis and DiI/DiO tracing of axons. Aim 2: The intermingling of OE and RE in the olfactory area and the occurrence of quiescent, aneuronal OE indicate that dysfunction of olfactory stem cells and progenitor cells contributes to peripheral olfactory pathology. Guided by our studies of the molecular profile of stem and progenitor cells in mouse and rat OE, we will identify markers for identifying neurocompetent stem and progenitor cells in human OE. We will screen for and analyze the expression of the human homologues of genes that are relevant in mouse using RT-PCR, IHC and in situ hybridization on human olfactory tissue collected by biopsy. We also plan open-ended gene profiling studies. The resulting panel can be used to assay for the stems and progenitors in human olfactory tissue. Aim 3: To test the usefulness of the stem/progenitor marker panel and the analysis of axon trajectory for the diagnosis of dysosmic patients. Guided by our studies of the olfactory area in Aim 1 we will harvest biopsies of the olfactory area from patients rendered dysosmic as a consequence of aging, chronic rhinosinusitis, or prior viral URI, and use our panel to analyze the status of stem and progenitor cells as well as the pattern of axonal growth within the tissue.

DESCRIPTION (provided by applicant): The capacity for regeneration of neuronal and non-neuronal populations in the olfactory epithelium (OE) is well-known and extends throughout life. By virtue of their accessibility, the neurocompetent stem and progenitor cells of the OE are attractive candidates for use in autologous cellular therapies. The regulatory signals and mechanisms that govern these processes are not well understood. An in-depth analysis of the regulation of olfactory epitheliopoeisis is needed in order to exploit the stem and progenitor cells fully, but progress has been slow. We have developed a tissue culture assay in which cells from the neonatal or adult lesioned OE grow within spheres that form at the air-media interface of culture well inserts in a condition- dependent manner. Remarkably, cells that are cultivated in 3-dimensions (3-D) retain the potency and plasticity to participate in the regeneration of the OE in the animal, which stands in sharp contrast to the failure of cells grown in 2-D to do so. Two Aims are proposed to explore the cues regulating cell generation and differentiation taking advantage of the in vivo-like properties of the sphere assay. Aim 1 will test how growth factors, alone or in combination, will affect the growth of OE cells in the 3-D cultures. Some of the factors have a previously established role in the turnover and regeneration process in the OE; they will calibrate the assay, providing registration of the in vitro and in vivo results. For the majority, little is known of their role in the OE, although they function elsewhere in cell birth or differentiation. In contrast, Aim 2 will determine the relevant cues within a complex molecular mix that is known to play an important role in regulating the OE. In this case, culture media conditioned by the growth of a lamina propria cell line (derived from the part of the mucosa that is deep to the OE) is required for the formation of spheres from adult lesioned OE on inserts. We will take both a candidate molecule and an open-ended approach to identifying the relevant factors. PUBLIC HEALTH RELEVANCE: The health relevance of the work resides in the ultimate goal of achieving regulated controlled growth of olfactory stem cells and progenitor cells. Besides the goal of helping patients with dysosmias or anosmias, the neurocompetent tissue stem cells may prove useful for repairing other parts of the nervous system, including the spinal cord after it is injured.

DESCRIPTION (provided by applicant): Abnormalities of stem cell function and/or the destruction of stem cells lead to an olfactory epithelium (OE) that remains olfactory (including a full apical lining composed of sustentacular cells), but one that has no neurons (OSNs), no globose basal cells (GBCs), and no ongoing neurogenesis - a condition that we term neurogenic exhaustion. We have developed a mouse model based on the expression of Diptheria toxin subunit A (DTA) in OMP-expressing mature OSNs that causes a tetracycline-reversible abbreviation of normal neuronal life-span and causes proliferation to accelerate at first, but then eventually dissipate. Aim 1 proposes experiments to understand when and how GBC stem cell failure develops by analyzing the GBC population as a function of age, as well as its capacity to generate olfactospheres in culture and engraft after transplantation - both of the latter phenomena assay the functional status of the progenitor population. Aim 2 shifts its focus to a second type of olfactory stem cell called horizontal basal cells (HBCs). Unlike GBCs, the HBCs persist in aneuronal OE, but are not functioning as multipotent progenitors/stem cells as they can do when the epithelium is directly injured and more than just neurons are dying. The HBCs will be activated out of their dormancy by a conditional knock-out strategy aimed at the transcription factor p63. We will test whether the GBCs that originate from the HBCs will persist and repopulate the epithelium. Aim 3 will assay whether restoration of the neuronal population, in this case by taking advantage of the reversibility of our DTA-driven experimental model and stopping the premature death of the neurons, results in correct reinnervation of the olfactory bulb. The experiments in Aim 3 will establish whether it is realistic to expect that the rejuvenation of the stem and progenitor populations and the reestablishment of neuronal input to the CNS can restore appropriate function. Currently, the complete lack of therapeutic options for patients with olfactory disease provides a strong justification for pursuing this research.

The capacity of the olfactory epithelium (OE) for replenishing the population of olfactory sensory neurons and for regenerating the epithelium after injury depends on the persistence and maintained function of stem cells within that adult epithelium. Decline in sensory function in the elderly is accompanied by pathological changes in the OE that emerge because the normally active olfactory stem and progenitor cells, namely globose basal cells (GBCs), become disordered and eventually depleted. At present, we can construct a flow diagram designed to encompass and sequence the various categories of GBCs, beginning with GBCs that function as multipotent progenitors, progressing through GBCs that act as transit amplifying progenitors, and reaching GBCs that give rise directly to neurons. We can also align those purported stages in the GBC hierarchy with the expression of various transcription factors. However, we lack a comprehensive understanding of this critical stem/progenitor cell population. Are these stages discrete or are they snapshots of a more fluid progression? How do we explain the plasticity in the progenitor capacity of some GBCs? How might aging disorder the progression? The current application proposes two specific aims designed to address the critical gaps in our knowledge of the active stem and progenitor cell population. Specific Aim 1 will generate transcriptomic profiles of the various functional categories of GBCs using transgenic mouse strains that express a fluorescent marker in conjunction with the transcription factor(s) gene(s) ? Sox2, Ascl1, and Neurog1 ? used to define the stages in the hierarchy. Specific Aim 2 will focus on and profile a specific kinetically- defined subset of GBCs that experience prolonged mitotic quiescence, as shown by the retention of the tagged histone fusion protein H2B/GFP, which is a feature common to many stem cell types. In both cases, cells labeled by expression of the fluorescent tags will be isolated by FACS, captured as single cells, and individually profiled. Analysis of the single cell expression libraries will be used for unbiased clustering of the cells and defining the genes whose expression differs across the clusters. The patterns of gene expression that differentiate the clusters will be validated by Q-PCR and either immunohistochemistry or in situ hybridization depending on antibody availability. At the conclusion of the analysis we will have achieved a comprehensive understanding of the GBC population and will clarify whether the existing flow diagram ? which envisions distinct stages and abrupt transitions ? is an accurate representation of the biology of this stem and progenitor cell-encompassing population. These advances, in turn, will inform our attempts to alleviate olfactory sensory dysfunction, particularly that which accompanies aging.

PROJECT SUMMARY The capacity of the olfactory epithelium (OE) for replenishing the population of olfactory sensory neurons and for regenerating the epithelium after injury depends on the persistence and maintained function of stem cells within that adult tissue. Decline in sensory function in the elderly is accompanied by pathological changes in the OE that emerge because the normally active olfactory stem and progenitor cells, namely globose basal cells (GBCs), become disordered and eventually depleted. In this setting, the reserve stem cells, namely the horizontal basal cells (HBCs), remain dormant despite the neurogenic exhaustion and disappearance of GBCs; in contrast, if the OE is damaged by an olfactotoxin, the HBCs activate and contribute to the repair of the epithelium. A therapeutic strategy that accomplishes controllable activation of HBCs in the setting of an exhausted OE offers possibly the best approach to treating age-related olfactory dysfunction. We have demonstrated that the transcription factor p63 is the master switch that regulates HBC activation ? a precipitous decline in p63 levels is necessary and sufficient for activation. Further, signaling by Notch1 maintains p63 levels and restrains activation; we hypothesize that the ligand for Notch1 is Jagged1 expressed by sustentacular cells, since their selective death is sufficient to activate HBCs. We propose 2 Aims in this application to build on previous advances. Aim 1 focuses on Notch signaling and asks how precisely do the complexities of the Notch pathway in the OE regulate HBCs? Additional questions address the other signals that derive from Sus cells to regulate HBCs. Finally, we will extend our studies manipulating Notch signaling in tissue culture to human HBCs. Aim 2 focuses on the activation process following injury and asks how does proteasomal degradation of p63 contribute to the decline in protein levels in mouse and in human HBCs? When completed, we will have achieved a much more thorough understanding of the process by which HBCs are shifted out of dormancy so that they might contribute to epithelial regeneration. That understanding of mechanism in both mouse, where genetic manipulations offer profound analytic power, and in humans will advance our efforts aimed at identifying therapeutic strategies for alleviating olfactory sensory dysfunction, particularly the sensory loss which accompanies aging.