Peter Beaumont Smith - US grants

Sympathetic innervation to Moller's smooth muscle in the superior eyelid of the rat normally is derived entirely from the ipsilateral sympathetic chain. Removal of the ipsilateral superior cervical ganglion (SCG) in juvenile and adult rats results in sustained SNS denervation and impairment of Moller's muscle adrenoceptor-mediated contractile response. In contrast, ganglionectomy in neonatal rats results in functional SNS reinnervation from the contralateral SCG. Contralateral reinnervation is beneficial, since contractile responses show less deficit and ptosis is markedly attenuated relative to older preparations in which reinnervation does not occur. The objectives of the present study are to characterize anatomical and physiological aspects of the contralateral projection, determine the cellular mechanisms responsible for enhanced neuroplasticity in neonatal rats and determine if contralkateral reinnervation occurs in other end organs. The specific aims of the project are to; 1) define the optimal age and time course for smooth muscle reinnervation, 2) determine the structural and functional correlates of contralateral reinnervation (LM histofluorescence and EM cytochemistry to assess density of innervation and neuro-muscular relationships; HRP transport to analyze numbers, size and distribution of ganglion cells; functional analysis of reflex contractile responses to selected stimuli), 3) determine effects on smooth muscle maturation using physiolgical/pharmacological methods to assess function and quantitative LM to analyze muscle volume and cell numbers, 4) determine effects of perturbations which may modify neonatal neuroplasticity, and 5) examine other end organs to determine if contralateral innervation following neonatal denervation is more widespread than realized. This study will attempt to ascertain the mechanism of enhanced neuroplasticity in the neonatal SNS which has not previously been studied. Furthermore, it will provide information of clinical relevance regarding possible amelioration or treatment of cervical SNS lesions (Horner's syndrome) for which adequate therapy does not exist.

DESCRIPTION: Peripheral nerve degeneration following injury has implications not only for the denervated target, but also for intact heterologous nerves. For example, sympathetic denervation causes changes in both structure and molecular phenotype of intact sensory and parasympathetic nerves projecting to a common target. Our preliminary studies show that heterologous nerve function can also be affected. Parasympathetic nerves innervating the rat superior tarsal muscle, an orbital smooth muscle, normally attenuate muscle contraction by inhibiting neurotransmission of excitatory sympathetic nerves. However, by 5 weeks after sympathectomy, parasympathetic nerves have become excitatory, eliciting a muscarinic cholinergic contraction; this is not attributable to muscle supersensitivity or diminished enzymatic degradation of acetylcholine, and is therefore believed to be due to changes occurring at the level of the nerve. Our objective is to determine mechanisms responsible for functional conversion, and its relevance to ameliorating deficits after nerve injury. The applicant hypothesizes that, as a result of sympathectomy, excitatory parasympathetic neuromuscular transmission is established because of increased neuroeffector contacts and/or increased transmitter production and release. The applicant further postulates that this occurs because of altered neurotrophic factor levels within the target, that it attenuates denervation- induced target deficits, and that it occurs in systems other than orbital smooth muscle. The specific aims are to further characterize parasympathetic functional conversion after sympathectomy by determining: (1) its time course, (2) if it is associated with more intimate neuromuscular contacts, (3) if changes occur in numbers of parasympathetic fibers, and (4) whether it is accompanied by increased activity of the acetylcholine-synthesizing enzyme, choline acetyltransferase. In addition, the applicant will determine (5) if it can be modulated by changing the availability of target-derived neurotrophic molecules, (6) if smooth muscle atrophy and supersensitivity are ameliorated, and (7) if functional conversion can be demonstrated in blood vessels of the eye and orbit. These studies investigate a novel and potentially important form of neuroplasticity. An understanding of this phenomenon will provide not only a clearer picture of its mechanism and prevalence, but also may give insight into how it can be manipulated to reverse denervation- induced target deficits, thus leading to improved medical rehabilitation strategies after nerve injury.

The objective of this application is to elucidate factors governing peripheral sympathetic nerve remodeling in the mature rodent uterus. Our published studies show that sympathetic nerve density of the virgin rat uterus fluctuates throughout the estrous cycle, a 4.5 day interval that is analogous to the human menstrual cycle. Nerve density is highest during diestrus and declines through estrus in association with rising plasma estrogen. Our preliminary experiments suggest that: 1) NGF mRNA and protein are also reduced at estrus, 2) estrogen administration decreases sympathetic nerve density, and 3) mice lacking a functional estrogen receptor alpha have uteri that are grossly hyperinnervated. We hypothesize that rising plasma estrogen suppresses uterine neurotrophic factor production resulting in clinical sympathetic nerve degeneration followed by regeneration. The specific aims are: 1) Characterize the structural changes in nerves occurring during the cyclical changes in uterine sympathetic innervation, 2) determine the estrous cycle hormonal factors that mediate changes in nerve density, 3) determine if uterine neurotrophic factors that induce sympathetic neuritogenesis vary during the estrous cycle 4) determine if exogenous estrogen or other hormones affect neurotrophin expression 5) determine if estrogen-mediated changes in sympathetic neuron neurotrophin receptor expression may also contribute to the neuroplasticity, 6) determine if antibodies that selectively block neurotrophin activity can prevent uterus-mediated sprouting in vitro, 7) determine the role of the estrogen receptor alpha on nerve and uterus using the ERKO mouse, and 8) assess the functional consequences of uterine sympathetic nerve remodeling. These aims will be accomplished using immunohistochemistry, in situ hybridization histochemistry, qualitative and quantitative RT-PCR, electron microscopy, enzyme-linked immunoassays, organ culture, and pharmacological analyses of neuroeffector transmission. These studies will provide new and important information on mechanisms underlying nerve remodeling in normal physiological conditions. This information may be important in advancing our understanding of the mechanisms regulating innervation in health and disease, and may have direct applicability to the understanding of dysmenorrhea and autonomic dysfunction that occurs in menopause.

DESCRIPTION (Adapted from Applicant's Description): Traumatic labor and vaginal delivery during childbirth can produce permanent dysfunction of the pelvic musculature, in many cases leading to urinary and fecal incontinence. Damage to the pelvic nerves and failure to achieve complete reinnervation account for much of the deficit. Factors that modulate regrowth of damaged axons therefore may influence functional recovery. The investigators have shown recently that smooth muscle of the reproductive tract, which shares many similarities with urethral and anal sphincter smooth muscle, undergoes dramatic changes in innervation as a consequence of hormonal fluctuations. Elevated plasma estrogen results in marked reductions in numbers of sympathetic nerves, while other neuronal populations are unaffected. Preliminary data suggest that these changes are related to decreased nerve growth factor (NGF) synthesis. The investigators hypothesize that the high levels of estrogen in periparous females result in depressed neurotrophin synthesis in pelvic smooth muscle. Accordingly, sympathetic nerves, whose presence is essential for normal sphincter contractile tone, fail to regenerate to their full potential after nerve injury. In Specific Aim 1, the investigators will determine the effects of estrogen and pregnancy on protein and mRNA levels of NGF and the related neurotrophin, NT3, in urethral and anal sphincter smooth muscle using in situ hybridization, quantitative competitive polymerase chain reaction, immunohistochemistry and enzyme-linked immunoassays. In the second aim, they will use quantitative in situ hybridization and immunohistochemistry to determine the extent to which estrogen and pregnancy influence expression of the neurotrophin receptors trkA and p75NTR, which mediate the sympathetic nerve response to NGF and NT3. In aim 3, they will use immunohistochemistry to examine the effects of estrogen and pregnancy on the normal innervation of the urethral and anal sphincter smooth muscles. Aim 4 will employ immunohistochemistry and physiological and pharmacological measurements of urethral and anal smooth muscle contractile function to assess the effects of estrogen on sphincter reinnervation following a noradrenergic neurotoxin lesion with 6-hydroxydopamine, or pelvic distension to simulate childbirth trauma, and these will be compared with injury of normal delivery. The fifth aim uses collagen gel co-cultures of sphincter smooth muscle and sympathetic ganglia in the presence of selective neutralizing antibodies to ascertain the roles of neurotrophins in modulating sympathetic neurite sprouting toward smooth muscle of estrogen-treated or pregnant rats. These studies should provide important new information on how hormones may affect neurotrophin synthesis by smooth muscle of the organs of continence, and how this in turn may alter sympathetic reinnervation of sphincters after axonal damage due to traumatic vaginal delivery, thus leading to urinary and fecal incontinence.

education evaluation /planning

DESCRIPTION (provided by applicant): Arrhythmias and cardiac dysfunction occur when sympathetic neural influences are excessive. Recent evidence indicates that structural remodeling of cardiac nerves contributes to hyperexcitability. Because nerve growth factor (NGF) is the major protein regulating sympathetic innervation, it is implicated in sympathetic neuroplasticity. In the present application we test the hypothesis that abnormal NGF synthesis after myocardial infarction leads to cardiac sympathetic nerve remodeling. We propose that abnormally high NGF expression in peri-infarct inflammatory myofibroblasts and macrophages contributes to hyperinnervation, and that deficient NGF expression in parasympathetic neurons leads to loss of parasympathetic presynaptic inhibition of sympathetic nerves. The long-term goals of this study are to understand the molecular mechanisms that regulate cardiac sympathetic innervation and to devise interventional strategies to correct abnormal innervation patterns. Experiments in Aim 1 demonstrate that coronary artery ligation in rats upregulates NGF in specific subsets of inflammatory cells. Aim 2 explores temporo-spatial features of ingrowth of identified axons and assesses the role of trophic factors in this process. Aim 3 investigates if sympathetic innervation itself promotes inflammatory cell NGF expression via beta adrenergic receptors. In Aim 4 we assess whether NGF expression in cardiac parasympathetic neurons, which may govern formation of axo-axonal inhibitory synapses, is regulated by sympathetic innervation. In Aim 5 we examine the role of adrenergic receptors in regulating cardiac parasympathetic NGF synthesis. Aim 6 investigates the possibility that adrenergic receptor down-regulation leads to diminished cardiac parasympathetic NGF synthesis in heart failure. Aim 7 investigates whether reduced parasympathetic NGF can account for diminished axo-axonal inhibition in heart failure. We use morphometric histochemistry, cell and tissue culture, protein and mRNA assays, and in vivo recordings to attain these goals. These studies will provide new and important information on molecular mechanisms regulating sympathetic neuroplasticity within the damaged heart, thus providing a more complete understanding of post-infarct cardiac dysfunction. Importantly, they will provide novel data on how trophic factor synthesis is regulated by adrenergic receptors, and may serve as a basis for pharmacological interventions aimed at preventing or reversing deleterious cardiac sympathetic remodeling.

[unreadable] DESCRIPTION (provided by applicant): Hormonal status and vaginal function are closely linked. Diminished reproductive hormones at menopause lead to vaginal atrophy and dryness. Menopause is often accompanied by dysesthetic vulvodynia, a pain syndrome consisting of burning and itching. Together with vulvar vestibulitis, an allodynia-like syndrome linked to early oral contraceptive use, vulvodynia represents an under-recognized but significant health problem, afflicting some 16% of the adult US female population. The etiology of these syndromes is poorly understood, although vulvar vestibulitis is associated with increased numbers of pain-sensing fibers. No animal models have been available to provide a better framework of understanding. Recently, we showed that estrogen regulates vaginal innervation in rats. Ovariectomy, which approximates human menopause, dramatically increases numbers of vaginal sensory nociceptors, as well as sympathetic and parasympathetic axons. We hypothesize that this is due to modulation of trophic factor release from vaginal tissues, and that altered innervation will influence key aspects of vaginal function, including blood flow, vascular permeability, and pain sensitivity. In aim 1 we propose to characterize the relationship between hormonal status and vaginal innervation in rats during the estrous cycle, pregnancy, and adult and juvenile hormone administration. We also determine if human vaginal innervation varies with hormonal state. Aim 2 assesses cellular mechanisms underlying axonal remodeling by determining effects of reproductive hormones on vaginal target tissue and on sensory and autonomic neurons. Aim 3 examines molecular mechanisms mediating vaginal remodeling by investigating expression and functional relevance of potential trophic factors. In aim 4, we assess the functional significance of vaginal nerve remodeling on blood flow, neurogenic inflammation and behavioral avoidance of painful stimuli. These studies are conducted using methods in cell biology, tissue culture, molecular biology, physiology, pharmacology and behavior. The findings of these experiments will provide insight into mechanisms underlying hormone-dependent remodeling of vaginal innervation, and whether altered innervation may contribute to vaginal dysfunction. Moreover, these studies will provide a better understanding of the relationship between vaginal nerve plasticity and vulvodynia, and potentially lead to new therapeutics aimed at reversing vaginal sensory hyperinnervation. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Remodeling of mature neural pathways involves both axonal outgrowth to establish new connections and axonal degeneration whereby terminations are eliminated. Mechanisms by which axon terminations are eliminated, or pruned, in the absence of cell death are poorly understood. Peripheral sympathetic innervation presents an especially tractable model for studying axon pruning under normal physiological and pathophysiological conditions. Sympathetic axon density in the virgin rodent uterus fluctuates rapidly during the estrous cycle, with terminal axons degenerating when estrogen levels rise and regenerating when they decline. We have shown that estrogen elevates uterine brain derived neurotrophic factor, and hypothesize that this contributes to sympathetic axon degeneration. We hypothesize that brain derived neurotrophic factor activates the p75 neurotrophin receptor, which stimulates intra-axonal ceramide formation. This promotes terminal axon degeneration through abnormal increases in membrane permeability and actin depolymerization. The present study investigates mechanisms whereby targets elicit selective terminal axon pruning. The first aim evaluates the hypothesis that p75NTR activation is responsible for inducing sympathetic axon degeneration under physiological conditions. In aim 2, we explore the hypothesis that p75NTR activation produces axon degeneration by increasing permeability of axonal membranes, and by promoting destabilization of the actin cytoskeleton. In aim 3, we investigate the nature of ligands produced by the target that incur axon degeneration. Specifically, the roles of BDNF, pro-NGF and neurotrimin will be assessed. These studies use tractable in vivo and in vitro approaches to explore relationships among target- derived ligands, neural receptors, and signal transduction pathways, and will provide novel information on how selective terminal axon degeneration is accomplished under physiological and pathophysiological conditions. Findings will be pertinent to understanding both organizing principles related to normal nervous system plasticity, and to disturbances in innervation in certain disease states.

[unreadable] DESCRIPTION (provided by applicant): Hormonal status and vaginal function are closely linked. Diminished reproductive hormones at menopause lead to vaginal atrophy and dryness. Menopause is often accompanied by dysesthetic vulvodynia, a pain syndrome consisting of burning and itching. Together with vulvar vestibulitis, an allodynia-like syndrome linked to early oral contraceptive use, vulvodynia represents an under-recognized but significant health problem, afflicting some 16% of the adult US female population. The etiology of these syndromes is poorly understood, although vulvar vestibulitis is associated with increased numbers of pain-sensing fibers. No animal models have been available to provide a better framework of understanding. Recently, we showed that estrogen regulates vaginal innervation in rats. Ovariectomy, which approximates human menopause, dramatically increases numbers of vaginal sensory nociceptors, as well as sympathetic and parasympathetic axons. We hypothesize that this is due to modulation of trophic factor release from vaginal tissues, and that altered innervation will influence key aspects of vaginal function, including blood flow, vascular permeability, and pain sensitivity. In aim 1 we propose to characterize the relationship between hormonal status and vaginal innervation in rats during the estrous cycle, pregnancy, and adult and juvenile hormone administration. We also determine if human vaginal innervation varies with hormonal state. Aim 2 assesses cellular mechanisms underlying axonal remodeling by determining effects of reproductive hormones on vaginal target tissue and on sensory and autonomic neurons. Aim 3 examines molecular mechanisms mediating vaginal remodeling by investigating expression and functional relevance of potential trophic factors. In aim 4, we assess the functional significance of vaginal nerve remodeling on blood flow, neurogenic inflammation and behavioral avoidance of painful stimuli. These studies are conducted using methods in cell biology, tissue culture, molecular biology, physiology, pharmacology and behavior. The findings of these experiments will provide insight into mechanisms underlying hormone-dependent remodeling of vaginal innervation, and whether altered innervation may contribute to vaginal dysfunction. Moreover, these studies will provide a better understanding of the relationship between vaginal nerve plasticity and vulvodynia, and potentially lead to new therapeutics aimed at reversing vaginal sensory hyperinnervation. [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The overall goal of the Outreach Core is to provide students on undergraduate campuses throughout Kansas with opportunities to become involved in and excited to pursue biomedical research as a profession. On these campuses, there is little specific focus;students major in biology, chemistry or, on some campuses, health related professions. Thus, outreach efforts are not specifically designed for trainees in cell and developmental biology. Programs described in the Progress Report and Undergraduate Support Core for the K-INBRE that we believe at this point to be successful are to be continued together with the Partnering Program, the purpose of which is to support travel and minor research related expenses for training, technology transfer, and collaboration among the Outreach Institutions (Haskell Indian Nations University, Washburn University, Fort Hays State University, Emporia State University, Pittsburg State University and Langston University) and Scientific Partner Hosts (KU Medical Center, KU Lawrence, Kansas State University, and Wichita State University) in specific areas of Cell and Developmental Biology.

[unreadable] DESCRIPTION (provided by applicant): Core Support for five years is requested for the competitive renewal of the Kansas Mental Retardation and Developmental Disabilities Research Center (MRDDRC). The Kansas MRDDRC, now in its 39th year, has played a major international role in generating highly effective behavioral interventions aimed at the causes, prevention, and treatment of mental retardation and related secondary conditions, and in delineating basic knowledge of the underlying biology of typical and atypical development. Since its inception, the center has supported a balanced portfolio of behavioral and biological research. Building on this rich history, a unique contribution of the center in the future will be the development of biologically informed behavioral and pharmacological intervention and treatment approaches. The mission of the Kansas MRDDRC is to support high quality basic and applied research relevant to the causes and prevention of mental retardation and the prevention and remediation of associated secondary conditions and related developmental disabilities. To achieve this mission, the Kansas MRDDRC is designed to accomplish three objectives. First, to develop and support new interdisciplinary basic and applied research initiatives directly relevant to the center's mission, bringing together scientists across the Kansas Center as well as promoting collaborative ventures with researchers at other institutions. Second, to provide cost-effective, scientifically generative, state of the art core services, resources, and facilities that directly enhance the quality and impact of science produced by center investigators and their collaborators. Third, to provide highly efficient, cost-effective systems for planning, developing, managing, coordinating, and disseminating research activities associated with the center. The Kansas MRDDRC's research program is organized around four integrated thematic areas that each reflects a general topic central to MRDD as well as the scientific directions and strengths of our current efforts. These themes are: 1) language, communication, and cognition of mental retardation; 2) risk, prevention, and intervention in mental retardation; 3) neurobiology of mental retardation; 4) cellular and molecular biology of early development. To coordinate and support the research activities of the 76 investigators and 75 research projects associated with these themes, four core units are proposed: a) Communication and Administration; b) Biobehavioral Measurement; c) Research Design and Analysis; d) Integrative Imaging. [unreadable] [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The Communications Core is established for the purpose of ensuring that the videonetwork linking the 10 Kansas INBRE campuses operates at the highest possible level of efficiency. It is the purpose of this core to offer a convenient and easily used system by which individuals and groups of researchers may speak together interactively or may view seminars and other educational experiences offered by different campuses. The Director of this Core will 1) have responsibility for overseeing all aspects of the Polycom communication system, 2) work to develop "deliverables" over this network that are of interest and importance to Kansas researchers on the 10 Kansas INBRE campuses, and 3) collaborate with the Bioinformatics and Administrative Core personnel as well as Kansas COBRE research administrators to ensure that news of program offerings is widely distributed and readily available.

DESCRIPTION (provided by applicant): Musculo-skeletal pain affects roughly half of the adult US population, resulting in enormous loss of productivity. Vitamin D deficiency is also extremely prevalent. Clinical studies suggest that populations prone to vitamin D deficiency, including the elderly and obese, are much more likely to experience muscle pain. Further, vitamin D repletion is reported to be effective in ameliorating muscle pain. However, the biological basis for hypovitaminosis D-induced pain is unclear. We've found that dorsal root ganglion sensory neurons express vitamin D receptors, and developed a rat model of vitamin D deficiency showing deep muscle sensitivity. Gastrocnemius muscle from these rats show increased indices of innervation, consistent with increased sensory axon density in a number of peripheral pain syndromes. The objective of this proposal is to explore the relation among vitamin D deficiency, sensory innervation and muscle pain. We hypothesize that vitamin D deficiency leads to sprouting of sensory nociceptor axons in skeletal muscle. In the first specific aim, we will characterize peripheral target innervation with regard to changes in innervation density, neuronal phenotype, and ability to activate spinal pathways in rats with normal or deficient vitamin D. In the second aim, we will determine direct effects of physiological and hypophysiological concentrations vitamin D on axon outgrowth from dissociated adult dorsal root ganglion neuronal cultures. We will also examine the role of target tissue by conducting explant cultures of ganglion and skeletal muscle from normal or vitamin D- deficient rats and assessing the muscle's ability to modulate neurite outgrowth. These studies represent the first systematic exploration of the biological basis of hypovitaminosis D-induced muscle pain. They are also the first to investigate a role of vitamin D in mature axonal outgrowth. Results from this exploratory study will provide a basis for additional mechanistic investigations using the rat model, and for clinical studies documenting the utility of vitamin D in preventing and reversing pain syndromes. PUBLIC HEALTH RELEVANCE: Both musculo-skeletal pain and vitamin D deficiency are widespread within the US population, and clinical studies suggest that a causal relationship exists. However, the biological basis by which vitamin D deficiency leads to muscle pain is unclear. We propose the first systematic investigation concerning cellular mechanisms by which vitamin D deficiency leads to muscle pain. These studies will provide an evidence-based rationale for employing vitamin D supplementation as a preventative or therapeutic approach to a problem that costs the US economy in excess of $40 billion per year.

DESCRIPTION (provided by applicant): An estimated 6 million women in the US suffer from vulvodynia. Provoked vestibulodynia, which typically is associated with vulvar vestibulitis, occurs most often in premenopausal women. This chronic pain syndrome is characterized by increased numbers of nociceptor axons usually localized to the posterior vestibule. Clinical evidence suggests that reproductive hormones influence the development and severity of vulvar vestibulitis syndrome (VVS). Aside from surgical excision of the hyperinnervated tissue, there are no effective therapies. This application proposes preclinical studies designed to characterize an animal model of VVS, and to use it to assess biological mechanisms that may be amenable to therapeutic targeting. We developed a rat model of VVS that replicates many clinical findings in humans. Small-volume injections of complete Freund's adjuvant into the rat posterior vestibule evoke persistent hypersensitivity and hyperinnervation. In Aim1, we use this model to investigate neural consequences of vestibular inflammation, including sprouting and phenotype alterations. We will investigate the persistence of hyperinnervation and its correlation to mechanical vestibular sensitivity, and determine if our model shows behavior consistent with dyspareunia. We will assess whether estrogen, which alters normal patterns of nociceptor innervation, also affects development of hyperinnervation. We will build on preliminary findings that progesterone administered to juvenile rats causes persistent increases in sensory innervation density, and determine whether this augments development of vestibular hyperinnervation. We will assess the extent to which our model simulates human cytological changes by comparing findings in rats with tissue excised from patients with VVS. In Aim 2, we test the hypothesis that activation of the angiotensin II receptor type 2 (AT2) mediates hyperinnervation and hypersensitivity in VVS. In preliminary studies, we show that AT2 blockade abrogates hyperinnervation and hypersensitivity in our model. We hypothesize that inflammatory cells create a local renin-angiotensin system that synthesizes angiotensin II, which initiates sensory axon sprouting. We will determine if angiotensin II is synthesized by rat and human vestibular tissue, and using explant cultures, that it elicits sprouting. We will determine i AT2 activation in the absence of inflammation elicits sprouting and hypersensitivity in our rat model. We will determine if AT2 antagonism not only prevents, but also reverses hyperinnervation and hypersensitivity. We will determine if AT2 blockade is overcomes hyperinnervation and mechanical sensitivity augmented by the actions of reproductive hormones. This application will provide fundamental information on mechanisms that regulate innervation in normal and inflamed vestibular tissue. It employs a novel rat model to identify the biological underpinnings of vestibular inflammatory hypersensitivity with the intention of manipulating a key signaling pathway in order to identify new therapeutic targets in VVS. Information obtained in these studies has strong potential to substantively change our thinking and clinical approach to the management of some forms of vulvodynia.

? DESCRIPTION (provided by applicant): High-throughput genome screening of patients provides hope for identifying biological mechanisms in rare, undiagnosed disorders, and may provide broader insight into more common disease categories. However, variant identification is only a first step; a cause-effect relationship between variant and phenotype must be established, and therapeutic strategy formulation requires a thorough understanding of cellular and protein pathologies. The NIH Undiagnosed Disease Program recently characterized a male patient, UDP1757 with neonatal onset of motor dysfunction, cognitive deficiencies, low brain volume, and peripheral neuropathy, worsening at 15 months, and who died at 10 years of age. Genetic screening showed a hemizygous single point mutation in the BHLHB9 locus (C318R) located on the X chromosome. BHLHB9 has been shown to prevent apoptosis and to promote neuronal differentiation and axonogenesis. We hypothesize that C318R results in a loss- or reduction-of-function mutation that results in increased neuronal cell death and reduced axon outgrowth, hence leading to cortical atrophy and broad-spectrum neural dysfunction. The objective of this proposal is to establish a role of the C318R mutation in abnormal neuronal death and reduced neuronal differentiation and axonogenesis. To do so, we propose in vitro assays involving modifications of the BHLHB9 gene in mouse neural stem cells and patient-derived, neutrally differentiated iPS cells in order to define cellular pathologies. We will additionally use mouse models in which Bhlhb9 mutations are introduced, behavioral phenotype determined, and CNS impact defined anatomically. We anticipate that the mouse will recapitulate the clinical phenotype of UDP1757, and that both cell assays and anatomical imaging will yield data consistent with our hypothesis that BHLHB9 C318R is a loss- or reduction-of-function mutation. A strength is a team of experts with complementary skills in pediatric genetics and rare diseases, genetic engineering, transgenic mouse and iPS cell technologies, rodent behavioral assessment, and neuronal culture and quantitation. Additionally, the proposal draws on the rich core resources of the Kansas Intellectual and Developmental Disabilities Research Center. This Exploratory/Developmental Research Grant Award will make significant contributions by establishing cellular processes responsible for an uncharacterized clinical phenotype, and will provide a basis for subsequent studies using the in vitro and in vivo tools developed in this study to formulate, screen and test therapeutic strategies to ameliorate this and related devastating developmental disorders.

Abstract Funds are requested to upgrade high-throughput genomic sequencing capabilities of the Genomics Core at the University of Kansas Medical Center through the purchase of an Illumina HiSeq 4000 sequencer. The Genomics Core (GC) has provided microarray services since 2002 and high throughput sequencing using the Illumina HiSeq 2000/2500 platform since 2011. Demand for our sequencing services has grown continuously since their inception, and we now provide services to a broad user base at the University of Kansas, affiliated Kansas Institutions, and throughout the Central Midwest region. Recent advances in sequencing technology have dramatically improved high throughput sequencing by reducing user cost, decreasing run time, and increasing the amount and quality of data; unfortunately, our current instrument is not able to take advantage of these key advances, with the result that the cost and quality of discovery by our investigators will suffer. The proposed upgrade to the Illumina HiSeq 4000 will ensure that our sequencing capabilities are and will remain optimal for the foreseeable future. The enhanced attributes of the HiSeq 4000 will be critical to supporting NIH-funded center grants that rely heavily on the Genomics Core, which include KUMC's Cancer Center, Alzheimer's Disease Center, Intellectual and Developmental Disabilities Research Center, Polycystic Kidney Disease Research Center, CTSA, and 3 Centers of Biomedical Research Excellence. The HiSeq 4000 will serve 28 major and 8 minor University of Kansas users with 31 (27 R01-equivalent) NIH grants, representing a subset of a larger user base at 25 institutions across a 14-state region. The HiSeq 4000 will be located in space already optimized for Illumina operations and will be integrated into a core network that provides comprehensive administrative infrastructure including online tracking of requests, automated billing, and user-polling for quality assurance, as well as bioinformatics and technical support and newly expanded IT infrastructure. New users are recruited using a proven, collaborative pilot program mechanism, and the GC is backed by a strong institutional commitment to both the acquisition and operation of the instrument. The acquisition of the Illumina HiSeq 4000 will enhance the rate and quality of scientific discovery of our large and diverse user group, but will also reduce costs to the investigator by approximately 57% per gigabase, thereby providing significant investigator savings that can be applied toward other NIH-funded research activities.