Jon E. Levine, Ph.D. - US grants

The central nervous system regulates gonadotropin secretion and maintains reproductive cycles through the intermittent release of gonadotropin-releasing hormone (GnRH). The direct study of this critical process has been limited by the lack of an effective method for measuring GnRH release in unanesthetized animals. Such a method - a push-pull perfusion technique - is now available and will be used to address two fundamental questions regarding the physiology of GnRH neurosecretion: What is the role of GnRH release in the regulation of gonadotropin secretion? What neural and endocrine factors are important in the physiological regulation of GnRH release? In the proposed studies, the release of GnRH will be monitored with the push-pull perfusion system, and gonadotropin levels will be measured in blood samples obtained concurrently from an indwelling jugular catheter. The role of GnRH in the stimulation of pulsatile LH release, pulsatile FSH release, the primary gonadotropin surge, and the secondary FSH surge will first be clarified. An analysis of the physiological regulation of GnRH by ovarian, pituitary, and hypothalamic factors will then be undertaken. Barbiturates will be used to determine if a 24 h neural clock regulates GnRH release, and if estrogen or progesterone can either amplify or block the expression of this daily neuronal signal. The effects of ovariectomy and acute estrogen treatment will also be assessed. In a second series of experiments, the "short-loop" feedback inhibition of GnRH release will be investigated through immunoneutralization of LH or the selective inhibition of FSH secretion by porcine follicular fluid. The hypothalamic regulation of GnRH by endogenous opioid peptide systems will then be characterized through systemic and local applications of opiate agonists and antagonists. The possible modulation of any opiate-GnRH interaction by estrogen, and the mediation of progesterone feedback effects by endogenous opiates will be examined. Finally, the existence of a GnRH-independent neural regulation of gonadotropin release will be investigated by means of push-pull trans-perfusion system. These studies will provide a substantial amount of new information pertinent to several long-standing questions regarding hypothalamic-pituitary-gonadal relationships. A greater knowledge of the neuroendocrine regulation of GnRH release should enhance the prospects of devising contraceptive methods based upon perturbations of hypothalamic/hypophysial function, and aid in treatment of reproductive endocrinopathies, such as hypothalamic amenorrhea.

The central nervous system regulates gonadotropin secretion and maintains reproductive cycles through pulsatile release of luteinizing hormone-releasing hormone (LHRH). The decapeptide is released into hypophysial portal vessels and conveyed to the anterior pituitary to regulate LH secretion. The amplitude and frequency of LHRH pulses are influenced, in turn, by feedback actions of circulating gonadal steroids. The objective of the proposed studies is to gain a more complete, integrated understanding of regulatory mechanisms which govern three critical functions of LHRH neurons - LHRH synthesis, LHRH release, and LHRH actions. In vivo radiolabeling will be used to estimate rates of LHRH synthesis, microdialysis will be utilized to measure in vivo LHRH release patterns, and an in vivo isolated pituitary paradigm will be employed to analyze physiological actions of LHRH. These methods will be used to test hypotheses that (1) ovarian steroids directly or indirectly accelerate LHRH biosynthesis, leading to an increase in LHRH pulse amplitude and initiation of the preovulatory LH surge, and (2) homeostatic suppression of LH secretion at other times is manifest, in part, by restraint of LHRH pulse frequency. Microdialysis will also be used to test the hypothesis that catecholamine and endogenous opioid peptide neurons mediate steroid feedback actions. A RCDA is requested to facilitate acquisition of the scientific and technical expertise required in the proposed studies. The applicant, Dr. Jon E. Levine, (Assistant Professor of Neurobiology and Physiology at Northwestern University) is a neuroendocrinologist whose outstanding research potential has already been established through innovative work in developing methods for study of in vivo neuropeptide release. With RCDA support, the candidate's teaching and administrative responsibilities will be reduced, enabling the following short-term career goals to be realized: (1) rapid progress in the proposed studies, (2) training and consultation in biochemical procedures with Dr. Jeffery McKelvy (Head, Neuroscience Research Division, Abbot Laboratories) and with Dr. Ka- Leung Ngai (Director, Biotechnology Center, Northwestern University) and (3) increased scientific interactions with reproductive biologist northwestern University. A long-term goal of the candidate is to fully understand the inter-and intracellular mechanisms by which neural and endocrine factors regulate LHRH neurons. Such knowledge may help in development of contraceptive strategies, and aid in treatment of reproductive disorders, such as hypothalamic amenorhea.

The purpose of this program is to train five predoctoral and three postdoctoral fellows in specific areas of reproductive biology, within the framework of an integrated, multi-disciplinary program offering a uniquely broad perspective of the reproductive sciences. The specific areas of training are: (i) Reproductive Endocrinology hormonal interactions among the hypothalamus, pituitary gland, and gonads, including cellular and molecular actions of neurohormones, gonadotropins, and gonadal hormones, and control of their secretions, (ii) control of Gametogenesis- cellular, molecular, and hormonal events involved in gametogenesis, with emphasis on developmental mechanisms, (iii) Pregnancy and parturition- molecular endocrinology and cell biology of the placenta, and (iv) Environmental Control of Reproductive Processes- neural mechanisms which mediate effects of environmental variables, such as photoperiod, on reproductive processes. Within these specific areas, a broad array of in vivo and in vitro methodologies are available from trainees, including the most current technologies for investigation at the molecular, cellular, and integrative levels. Our nine preceptors are from four departments at Northwestern University, and each is currently funded by NICHD grants, including 10 R01 grants, a program project grant, and a Center for Research on Fertility and Infertility. Predoctoral trainees are selected from large, highly qualified pools of graduate students who complete at least one year of study within the departments and programs of the preceptors. Postdoctoral fellows are likewise chosen from an excellent application pool; virtually all postdoctoral trainees supported initially on this training grant successfully compete for NRSA support in subsequent years. Preceptors in this program have established a strong history of collaboration in their research this spirit of interaction continues to permeate the pre- and postdoctoral training arenas. Fellows participate in laboratory research, didactic courses, a class on ethical scientific conduct, research seminars, journal clubs, a yearly Symposium on Reproductive Biology, and meetings devoted to important professional issues. As a result, our trainees receive intensive training in specific area of reproductive research, acquire a broad perspective of reproductive biology as a whole, and are exposed to an environment of interdisciplinary collaboration. Through administration of this program, we have continued to produce highly-trained, successful scientists who carry with them a continuing interest in an integrated, multi-level approach to the study of reproductive biology.

high performance liquid chromatography; neurotransmitters; radioimmunoassay; hormones; biomedical facility; bioassay;

Gonadotropin surges are critically important biological events that depend upon the appropriate cellular integration of ovarian and neuroendocrine signals. Primary LH and FSH surges are dependent upon integration of neural and hormonal (estrogen, progesterone) stimuli in the brain, and secondary FSH surges require coordinated actions of ovarian steroids, inhibin, and activin in pituitary gonadotropes. We recently obtained evidence that stimulation of both the preovulatory and secondary gonadotropin surges requires activation of progesterone (P) receptors (PRs), even in the absence of circulating P. We have thus proposed that signals leading to release of GnRH surges from hypothalamus, and to secondary FSH surges from pituitary, must converge at the level of PR in neurons and gonadotropes, respectively. We hypothesize that these integrative processes depend upon E/2,s capacity to induce PR expression, and the ability of neuroendocrine signals to activate these PRs in a ligand-independent manner. Specifically, we will test the idea that neurotransmitter signals can activate neuronal PRs, leading to stimulation of GnRH and primary gonadotropin surges, and that activin and P can activate PRs, contributing to release to release of secondary FSH surges. In Aim 1, we will use a microdialysis approach to determine if contributing to release of secondary FSH surges. In Aim 1, we will use a microdialysis approach to determine if GnRH surges in steroid-treated ovariectomized (OVX) rats are blocked by pretreatment with P antagonists,or i.c.v. infusions of PR antisense oligonucleotides to disrupt PR synthesis. In Aims 2 and 3, we will use a gene transfer method to introduce a fusion gene containing a progesterone response element (PRE) linked to a reporter gene, into hypothalamic neurons in vitro and in vivo. We will use this approach to directly determine ligand-independent activation of PRs occurs in central neurons, and ascertain whether this process is associated with the preovulatory surge-generating process. In Aim 4, we will directly assess the involvement of the PR in mediating the effects of activin on FSH secretion. Activin stimuli will be presented to anterior pituitary cell cultures derived from wild-type and progesterone receptor knock-out (PRKO) mice, and FSH responses will be measured and compared. If a response deficit is noted in the PRKO-derived cultures, then we will conduct gene transfer experiments in which we will introduce the mPR into PRKO-derived cells, and determine the ability of these treatments to rescue FSH responsiveness to activin. In Aims 5 & 6, we will transfer the PRE-reporter fusion gene into anterior pituitary cells both in vitro and in vivo to determine if activin can activate PRs, and if this process is indeed associated with the estrous release of FSH. The information gained in these studies will be of major significance in our understanding of the integrative, cellular, and molecular events that comprise the periovulatory surge-generating process. As such, it could have important implications for the understanding of infertilities associated with disruptions of steroid feedback and production of mid- cycle ovulatory hormone surges. Of broader significance, these experiments may also may also provide new insights into the cellular mechanisms which function to integrate endocrine and neural signals, and thereby bring about a variety of biological responses, such as those involved in motivated behaviors, reproductive development, stress responses, or learning and memory.

DESCRIPTION: (provided by the applicant) The nervous system regulates the reproductive axis through the activity of the "GnRH pulse generator," neurons in the hypothalamus whose activity governs pulsatile release of gonadotropin-releasing hormone (GnRH). The amplitude and frequency of GnRH pulses are, in turn, regulated by physiological signals, some of which evoke sustained shifts in the activity of the GnRH pulse generator and hence, in overall reproductive state. Metabolic cues, for example, are clearly important for maintenance of GnRH pulsatility in adult animals, and in the deceleration of GnRH pulsatility that occurs under conditions of negative energy balance. Cellular and molecular mechanisms mediating physiological shifts in GnRH pulse generator activity remain unknown. These studies will test the hypothesis that metabolically-linked adjustments of GnRH pulse generator activity are mediated by regulation of ion channels, specifically those known to link cell metabolism to cell excitability - the ATP-sensitive potassium channels (K+ATP channels). The K+ATP channel closes upon binding AlP, leading to reduced cellular K+ permeability, membrane depolarization and thus, increased cell excitability; reduced intracellular ATP produces the opposite conditions, reducing cell excitability. Preliminary work implicates hypothalamic K+ATP channels activity in the modulatation of GnRH release. The proposed studies are therefore designed to determine whether K+ATP channel opening and closing in vivo lead to alterations in GnRH pulse generator activity (Aim 1), assess whether opening of K+ATP channels mediates inhibition of GnRH pulsatility during food-restriction (Aim 2), determine the molecular and functional properties of KATP channels in identified GnRH neurons [Aims 3,4], and determine whether suppression of hypothalamic K+ATP channel expression reverses effects of negative energy balance on GnRH pulsatility (Aim5) A transgenic mouse will also be developed (Aim 6) in which expressesion of overactive (open) K+ATP channel subunits will be targeted to GnRH neurons, permitting examination of the reproductive consequences of K+ATP channel hyperactivity in GnRH neurons. These studies may provide new and important information on cellular mechanisms mediating many major physiological alterations in GnRH pulse generator activity. They may also permit an understanding of how GnRH pulse generation is inhibited in feeding disorders such as anorexia nervosa, or in other hypogonadotropic states, such as functional hypothalamic amenorrhea associated with subclinical eating disorders or rigorous exercise training.

[unreadable] DESCRIPTION (provided by applicant): The hot flash, an abrupt sensation of heat, is the most common medical complaint among peri- and post-menopausal women. Peripherally, hot flashes are characterized by vasodilation, increased heart rate, and perspiration. The expression of the hot flash is correlated with lowered levels of circulating estrogen at the onset of menopause or surgical removal of the ovaries. Estrogen receptors are widely distributed throughout the central nervous system and the cardiovascular system. Within the central nervous system estrogen receptors are prominently expressed in the preoptic area (POA) of the hypothalamus - the location of temperature sensitive neurons associated with thermoregulation. It is thought that hot flashes are caused by a disturbance of normal thermoregulation brought on by the loss of estrogen; however, the underlying molecular, cellular, and physiological mechanism is unknown. Recently, a new family of ion channels, the background leak potassium channels or two-pore domain potassium (K2P) channels has been identified through the use of bioinformatics. In cell and tissue culture systems these channels have been shown to contribute the establishment of the resting membrane potential of cells. Activation of K2P channels causes hyperpolarization of cells resulting in inhibition of firing. Although some of the channels have been associated with temperature sensitivity, sensitivity to pain, response to inhalant anesthetics, and neuroprotection, the physiological functions of most of these channels in living organisms have yet to be established. It is hypothesized herein that estrogen regulates the expression of K2P channels in the central nervous system and that the withdrawal of estrogen results in a decrease in the number or activity of these channels. This reduction would depolarize neurons leading to greater excitability and the induction of hot flashes. The work proposed in this application will use a functional bioassay, radiotelemetric monitoring of tail skin temperature from estrogen deficient and replete mice, to monitor the effects of drugs that activate and inhibit K2P channels in a whole animal model. Immunohistochemistry will be used to determine whether or not the proteins are expressed in the same cells as estrogen receptors. Expression of the messenger RNAs for the channels will be quantified using real time-PCR and in situ hybridization. Although this application will investigate the role of K2P channels in generating hot flashes, increasing our understanding of the physiological functions of these channels may provide insight into mechanisms of other age-associated diseases associated with hyperexcitability of the central nervous system. Hot flashes are the most common medical complaint among peri- and post-menopausal women. Currently, the most commonly used treatment to alleviate hot flash symptoms is hormone replacement therapy (HRT). Unfortunately, HRT is associated with several risks, including an increase in hormone dependent cancers, leading many to seek other treatments. The cellular and molecular mechanisms that cause hot flashes are unknown. The work proposed in this application is designed to investigate the underlying cellular and molecular mechanisms responsible for hot flashes, leading to the development of safe and effective treatments. [unreadable] [unreadable] [unreadable]

Ovarian estrogens exert critically important homeostatic feedback actions in the brain, pituitary, and ovary to restrain androgen biosynthesis. Disruption of these feedback controls invariably leads to dysregulation of gonadotropin secretions, androgen excess, and impaired fertility. Estrogen receptor alpha (ERa) appears to mediate most of these major negative feedback actions. ERa signaling mechanisms include "classical genotropic" effects mediated by direct binding of receptors to DNA, "non-classical genotropic" effects involving tethering of ERs to other transcription factors, and "non-classical non-genotropic" actions mediated by ERs coupled to membrane-initiated signaling pathways. The proposed studies use novel ERa mutant mice to further determine cellular mechanisms by which ERa mediates E2 feedback effects. We have utilized mutant ERa knock-in mice, which confer non-classical genotropic and non-genotropic signaling in the absence of classical signaling, to determine that non-classical ERa signaling conveys E2 negative feedback actions on LH secretion. Aim 1 determines if these feedback actions are manifest in brain by directly monitoring GnRH secretory responses to E2 treatments in WT (ERa+/+), ERa gene knockout (ER-/-), and non-classical ERa gene "knock-in" (ER-/AA) mice, as well as in neuron-specific ERa-/- mice. Aim 2 examines the involvement of non-classical genotropic vs. non-genotropic ERa signaling in negative feedback;the ability of E2 to exert actions in afferent circuitries controlling GnRH release will be compared among the three genotypes. Aim 3 analyzes the locus of intraovarian feed-back in theca cell-specific ERa null mutant mice. We then assess involvement of classical vs. non-classical ERa signaling in regulating androgen synthesis by analyzing E2 effects in cultured ovarian follicles of ERa+/+, ERa/-., ERa-/AA mice. In Aim 4, we determine the extent to which E2 actions are mediated by non-classical genotropic vs. membrane-initiated ERa signaling. New animals will be generated that express ERs conferring non-classical membrane-initiated signaling only, or non-classical genotropic signaling only. The extent to which E2 actions in the reproductive axis are rescued in these mice will be determined. These experiments will clarify the basic mechanisms by which ERa signaling regulates cellular and physiological function, and may provide important insights into the pathogenesis of hyperandrogenism in endocrine disorders such as polycystic ovary syndrome (PCOS).

Project Summary The central nervous system regulates gonadotropin secretion through neurosecretion of gonadotropin- releasing hormone (GnRH). GnRH is synthesized in preoptic and periventricular neurons, transported via intertwined processes that distribute as axonal-vascular terminals in the median eminence and released into the hypothalamic-hypophysial portal vasculature. GnRH binds receptors on gonadotropes to stimulate secretion and synthesis of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Neurosecretion of GnRH is pulsatile and is obligatory for sustaining normal gonadotropin secretion and synthesis. Kisspeptin (KISS1) neurons are a major GnRH afferent network that activates GnRH neurons during puberty, maintains GnRH release during adulthood, and transduces negative and positive feedback actions of gonadal steroids. In our studies we will test the hypothesis that KISS1 neurons, specifically those in the arcuate nucleus (ARC) that co-express the peptides neurokinin B (NKB) and prodynorphin (PDyn) (KNDy neurons), function as a GnRH pulse generator (Aim 1), with NKB mediating synchronized activation of KNDy cells (Aim 2) and dynorphin terminating each pulse discharge (Aim 3). These studies are significant because they will provide information on the cellular circuitries that govern GnRH pulse generation, and thus will shed new light on the pathophysiologic mechanisms underlying neuroendocrine-based fertility disorders, such as absent, delayed, or precocious puberty, functional hypothalamic amenorrhea, or polycystic ovary syndrome, and in the longer term, point to new potential targets for intervention in these reproductive impairments. The proposed studies will use powerful genetic tools to dissect and characterize the functional components of the kisspeptin-GnRH pulse generating system. To define the key elements of the pulse generator, we will utilize innovative genetically modified mouse models, each enabling an analysis of the consequences of kisspeptin neuron-specific gene deletion. We will first study the role of the KNDy neuronal population using a Kiss1fl/fl mouse recently created in our laboratory. This mouse will be bred to a prodynorphin Cre (Pdyn-IRES- Cre21,2) mouse, hereafter Pdyn-Cre, to eliminate Kiss1 in KNDy neurons. We have also developed a novel mouse bearing a doxycycline-inducible kisspeptin-Cre allele (iKiss-Cre mouse) that will enable temporal control over the selective deletion of genes from Kiss1 neurons. Two other lines, a NKBfl/fl mouse (developed in our laboratory) and an Oprk1fl/fl mouse (Jackson labs) will be crossed to Kiss1-Cre or iKiss-Cre mice to generate mice that will allow us to analyze the effects of kisspeptin neuron-specific deletion of NKB and Oprk1 on GnRH- triggered LH pulses. We will also use the iKiss-Cre mouse to delete and study the roles of steroid receptors in kisspeptin neurons in the adult. This temporally controlled gene deletion will eliminate a longstanding technical limitation of conventional steroid receptor knockout models in which steroid regulation of the axis is confounded by steroid developmental and organizational effects in the axis.

Hyperandrogenism is a core feature of polycystic ovary syndrome (PCOS). PCOS is associated with reproductive abnormalities that include accelerated gonadotropin-releasing hormone (GnRH) release, LH excess, oligo- or amenorrhea, arrested ovarian follicular maturation, and development of follicular cysts. Clinical and animal studies have provided evidence that hyperandrogenism contributes to the manifestation of these abnormalities by inducing resistance to steroid negative feedback of pulsatile GnRH release. Little is known, however, about the neuronal mechanisms, including steroid receptors, signaling pathways, and neuronal populations, that mediate the negative feedback actions of estradiol and progesterone in the hypothalamus of female primates. Moreover, the pathogenic mechanisms by which androgens impair neuronal responsiveness to estrogen and progesterone feedback remain obscure. Project II studies will make use of viral vector-mediated gene silencing and a validated nonhuman primate model of androgen-induced reproductive PCOS phenotypes to address these major gaps in our understanding of the mechanisms that mediate the pathogenesis of PCOS. Using these methods, we will directly test the hypotheses that 1) estrogen receptor alpha (ERa) in hypothalamic arcuate nucleus (ARC) neurons mediates physiological negative feedback in the rhesus macaque, 2) androgen excess attenuates estrogen-induced-expression of progesterone receptors A and B (PR A/B) and confers resistance to negative feedback actions of estradiol and progesterone, and 3) reduced PR A/B expression in the ARC recapitulates the reproductive abnormalities of PCOS. These studies will thereby establish the normal physiological receptors and target cells that mediate steroid negative feedback in a primate, identify androgen-induced resistance to estradiol and progesterone actions as a fundamental pathological mechanism underlying PCOS-like reproductive dysfunction, and directly test the validity of the concept that hypothalamic resistance to progesterone is a determinant of reproductive abnormalities in PCOS.

? DESCRIPTION (provided by applicant): Juvenile development is characterized by pronounced suppression of reproductive hormone release, a mechanism that maintains gonadal quiescence until the onset of reproductive maturation. In primates, this juvenile brake is believed to be mediated by signals that arise within the brain and actively suppress hypothalamic neurosecretion of the major neural effector of reproductive hormone release, gonadotropin- releasing hormone (GnRH). Diminishment of this central restraint is believed to contribute to the acceleration of pulsatile GnRH release that, in turn, initiates pubertal maturation. Premature lifting of this restraint may underlie the pathogenesis of true precocious puberty, as well as earl pubertal onset within the range of normal variation. The neural signals that impose the juvenile brake remain unclear. In adulthood, the GnRH pulse generator is homeostatically suppressed by negative feedback actions of gonadal steroids, principally estradiol in females. Recent studies have demonstrated that estrogens produced in the brain itself may modulate GnRH release, and we have obtained preliminary evidence to suggest that extra-gonadal estrogens may contribute to the suppression of the GnRH pulse generator. The proposed studies are therefore designed to test the novel hypothesis that neuroestrogens mediate the prepubertal restraint of GnRH release during the juvenile period of development in female primates. In Aim 1, prepubertal female rhesus macaques will be ovariectomized and treated with a vehicle or letrazole, an inhibitor of the estrogen synthesizing enzyme, aromatase (CYP19A1). We will monitor secretion of pituitary gonadotropins, LH and FSH, to determine if systemic inhibition of aromatase in non-gonadal tissues results in a premature activation of GnRH release. In Aim 2, a viral vector expressing shRNA corresponding to aromatase, or a vector expressing a scrambled RNA sequence, will be injected into the hypothalamic areas critical for negative feedback regulation of GnRH release. Gonadotropin levels will be monitored to determine if localized hypothalamic inhibition of aromatase expression likewise results in premature activation of GnRH release. We predict that the results obtained in Aim 1 will demonstrate that non-gonadal estrogens exert some or all of the juvenile restraint on the GnRH pulse generator, and that Aim 2 will confirm neuroestrogens as specific mediators of this important physiological process.

Estradiol (E2) in adult female rodents regulates body weight, adiposity, energy balance, physical activity and glucose homeostasis, as demonstrated by robust metabolic responses to ovariectomy, E2 replacement, and genetic manipulation of estrogen receptors. In female primates, however, removal of the ovaries does not reliably lead to increases in adiposity, insulin resistance or altered energy homeostasis. In recent studies, we have confirmed that neither ablation of peripheral E2 production nor E2 replacement alters adiposity, glucoregulation or energy homeostasis in marmoset monkeys, adding to previous evidence suggesting peripheral estrogens do not play important roles in female primate metabolic regulation. Our recent novel findings, however, demonstrate the critical importance of hypothalamic estrogen receptor alpha (ER?) in regulating metabolic function in a female nonhuman primate (NHP), as silencing of hypothalamic ER? gene expression induces obesity and insulin resistance in adult female rhesus monkeys. Taken together, these findings suggest that hypothalamic ERa may function in NHPs, as it does in rodents, to regulate adiposity, glucoregulation and energy homeostasis, yet peripherally produced E2 has diminished importance in engaging these actions. Recent studies have suggested that neurosteroidogenesis, specifically hypothalamic aromatization by CYP19A1 of androgen precursors to E2, may regulate neural control of metabolic function in NHPs. We have therefore formulated a new hypothesis that E2 synthesized in the brain, specifically in neurons of the ventromedial nucleus (VMN) and arcuate nucleus (ARC) of the hypothalamus, activates ER? to regulate adiposity, glucoregulation and energy homeostasis in female NHPs. To test this hypothesis, we will use both pharmacological and viral vector- mediated shRNA approaches to determine if adiposity, glucoregulation and energy metabolism are altered by inhibition of the CYP19A1 aromatase enzyme, or by permanent silencing of the CYP19A1 gene in the hypothalamus of female rhesus macaques. We will also analyze the synthesis of E2 in the VMN and ARC by a microdialysis approach, and determine whether VMN and ARC E2 originates from hypothalamically synthesized androgens. These studies may fundamentally change our understanding of metabolic control of adiposity, glucoregulation and energy homeostasis by sex steroids in female NHPs, and prompt exploration of new therapeutic strategies to diminish metabolic disease in women.

PROJECT SUMMARY/ABSTRACT The common marmoset (Callithrix jacchus) has emerged as a critically important and tractable non-human primate (NHP) model for neuroscience research accommodating genetic manipulation and directed breeding. Several barriers to the adoption of marmoset models by the neuroscience community exist, including a small census size in the United States (fewer than 2,500 animals), poor communication about resource availability, a poor understanding of the strengths and limitations of marmosets in research, and a lack of a formal structure for coordinating the sharing of information and resources related to marmoset research. In this application, we propose to establish a Marmoset Coordinating Center (MCC) as part of the NIH Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative that will address these limitations by building upon our experience in NHP informatics and marmoset research. As the foundation for the MCC, we will build a new informatics framework for coordinating marmoset research based on the LabKey data management system. This infrastructure will be derived from the existing LabKey system in place at multiple National Primate Research Centers, and which has been optimized for NHP research, including marmosets, over years of production. This informatics system will be initialized with basic statistics on the marmoset laboratory populations provided by participating centers, making pre-recorded, individual-level information that is already recorded more broadly available to the neuroscience community. Demographic and genetic information will be shared and discussed among participating centers, to coordinate marmoset colony management in line with American Zoological Association practice guidelines. Once operational, the MCC will be comprised of two parts: 1) a web-based portal to provide access to the marmoset data resources stored in LabKey, as well as background content on marmoset biology and neuroscience; 2) a concierge comprised of MCC staff and augmented by a scientific advisory board of marmoset neuroscience experts. The website will provide summaries of existing marmoset neuroscience projects, and allow interested investigators to search animal inventory and request animals. The concierge will be available to answer basic questions about the Center and the use of marmosets in neuroscience. Together, the data and human resources of the MCC will provide an essential portal into marmoset neuroscience research through collaboration, service agreement, or animal transfer.

Summary Demand for the common marmoset (Callithrix jacchus) in biomedical research has increased tremendously over the past five years, as they have emerged as a critical biomedical model system in a variety of study disciplines. The increased use of marmosets has been most acute in neuroscience, where the need to study cognition, behavior, and mental illness in primate models has grown. Among non-human primate (NHP) models, the marmoset provides unique practical advantages for neuroscientific studies, including small size, easy handling, rapid reproductive maturation, high fecundity, compressed life cycle, and social behaviors and communication that more closely resemble those observed in humans. These advantages have also contributed to the recent surge in the demand for marmosets in studies using embryonic new genomic editing techniques. This recent upsurge in demand has resulted in shortages in availability of marmosets estimated to be in the range of 2000 marmosets needed by the overall research community each year, with our own estimate in the range of a 200- animal shortfall for the neuroscience community alone. The Southwest National Primate Research Center (SNPRC) and the Wisconsin National Primate Research Center (WNPRC) propose a major collaborative effort to dramatically increase the availability of healthy common marmosets for national neuroscience research projects. In response to the upsurge in demand for marmosets in neuroscience-related projects, the SNPRC and WNPRC propose a comprehensive plan to 1) double their output of marmosets for provision to national institutions in need of these experimental animals for neuroscience research, 2) continue to ensure the highest standards of husbandry and veterinary care and thus, the excellent health of these colonies, 3) maintain pedigree records and develop genomic databases to document and maximize genetic diversity among the expanded marmoset colonies, 4) continue collaboration between the two Centers in new studies focused on improving husbandry, breeding, and veterinary practices to maximize the health and lifespan of these animals, and 5) communicate on a regular basis with the U24 Marmoset Colony Coordination Center, and with the NIH Marmosets for Neuroscience Steering Committee. The collaborative approach proposed by the two NPRCs is both innovative and appropriate given the resources and expertise available and ensures successful completion of these aims resulting in a dramatic increase in the number of marmosets available to the neuroscience community.

Project Summary: CRISPR/Cas9 is a revolutionary and versatile genome editing technique with wide-ranging utility. In vivo genome editing is anticipated to be the next wave of therapeutics for various major health threats, including neurode- generative diseases. However, there is an urgent need to develop efficient, non-viral delivery vehicles for safe and efficient in vivo CRISPR genome editing. Furthermore, delivering CRISPR genome editing machinery to the brain/neuron represents a major hurdle due to its dense structure and the blood?brain barrier (BBB). The objective of this project is to engineer a family of versatile, novel, non-viral Cas9-gRNA ribonucleoprotein (RNP) delivery nanocapsules (NCs) that can robustly and safely generate targeted gene edits in neurons within the brain. We envision that our robust and universal RNP delivery nanoplatforms will enable innovative treatments for devastating neurodegenerative diseases. Towards this goal, we will evaluate the feasibility of our approach, in a demonstration, by targeting the amyloid precursor protein (APP) ? relevant to Alzheimer's disease (AD) in healthy mice and monkey models. During our preliminary studies, we developed a PEGylated NC with a high RNP loading content (68 wt.%), versatile surface chemistry, ultrasmall size (dH~13 nm), controllable stoichiometry, excellent biocompatibility, and high genome editing efficiency in vitro and in vivo. In UG3 Aim 1, we will further optimize the design of the NC for brain/neuron-targeted genome editing. In particular, we will investigate the synergistic effects of hybrid targeting ligands, including (1) glucose+RVG peptide for intravenous (i.v.) injection to enhance the crossing of the BBB and neuron-specific editing, and (2) CPP+RVG peptide for intracerebral injection to enhance uptake and neuron-specific genome editing. The effects of different types/amounts of targeting ligands on the cellular uptake, biocompatibility, genome editing efficiency, and functional consequences of the NCs in both Neuro2a and primary neuron cells will be investigated. In UG3 Aim 2, we will evaluate the brain/neuron targeting specificity, genome editing efficiency, and potential immune response and systemic toxicity of the i.v. or intracerebrally administered NCs conjugated with various targeting ligands in healthy mice. In UH3 Aim 1, we will develop the set up and synthesis process to scale up the production of NCs. In UH3 Aim 2, we will further evaluate the genome editing efficiency and biocompatibility of the brain/neuron-targeted NCs in healthy rhesus macaques. Our uniquely designed NCs are expected to achieve high brain accumulation, high penetration depth, and high neuron-specific genome editing efficiency due to their desirable characteristics. Given the modularity and ease of targeting different genes by the CRISPR system, we anticipate that the resulting NCs will be useful for a wide range of human diseases, including debilitating neurodegenerative diseases for which there are no cures.

Hyperandrogenism lies at the pathogenic core of polycystic ovary syndrome (PCOS). PCOS is a highly heritable trait (heritability = 70%) affecting >5 million reproductive age women in the USA, alone. Consequently, PCOS is a costly (~$14 billion/year) women?s health disorder, bestowing infertility, irregular menstrual cycles, polycystic ovaries and a complex array of cardiometabolic and malignant diseases. Progress towards prevention has been hindered by poor understanding of the pathogenic mechanism(s) and complete lack of naturally occurring animal models. Our recent discoveries, however, provide a unique opportunity to break through this impasse. A subset of female Indian rhesus macaques at the Wisconsin National Primate Research Center exhibit naturally occurring hyperandrogenemia (High testosterone, T) accompanied by core features of PCOS: infertility, LH and AMH excess, and insulin resistance. Together with our previous discovery that female macaques exposed in utero to T exhibit PCOS-related traits, these naturally High T macaques provide strong evidence that endogenous female hyperandrogenism programs PCOS-related pathology. In preliminary studies, we employed whole genome sequencing of High T rhesus macaques and contemporary controls and uncovered likely functional missense variants in High T females within previously identified human PCOS candidate genes. Our preliminary findings suggest conservation of genetic pathways and causal mechanisms accompanying hyperandrogenism in female macaques that have the potential to elucidate pathogenic mechanisms. We thus propose to evaluate pedigrees of genealogical female relatives and descendants of High T female Indian rhesus macaques to examine the hypotheses that (1) such relatives represent a population enriched with PCOS-related phenotypes, including but not limited to High T, (2) natural T levels are heritable, and (3) a survey of PCOS candidate genes will illustrate potentially functional variants in High T females. The goal of this R21 proposal is to evaluate the feasibility of developing an Indian rhesus macaque model of PCOS that is both phenotypically relevant to the human disease and results from similar underlying genetic mechanisms. While our prior studies have clearly demonstrated important similarities between High T phenotypes in female rhesus macaques and equivalent traits in women, we cannot at this point document either the heritability of High T in female macaques or the array of mutations segregating among these macaques within 26 known risk genes associated with PCOS in women. No full-scale genetic analysis of the Indian macaque model is warranted without prior evidence of both genetic influences on High T and the potential for parallel genetic mechanisms. This R21 project seeks to determine if these known dimensions of human PCOS are evident in a macaque model of naturally occurring, PCOS-related phenotypes, thereby confirming its unique suitability to acquire profound new insights on the genetic and pathophysiological bases of this reproductive and metabolic disorder.

Abstract. The rate of progress in understanding the basis for reproductive health and disease is breathtaking, accelerated ever further by constant advances in cellular and molecular techniques. Nonetheless, research in traditional academic departments and research in clinical departments often does not overlap. As such, the PhD trained in a purely academic environment will remain unexposed to the clinical mindset and practices and so fail to pursue translational goals, while the clinician Fellow is well aware of the clinical problem and by definition focused on the human itself, but may be less aware of recent conceptual and methodologic advances available to investigate clinically derived research questions. As PA-18-403 RFA states, ?past studies have shown that health professional trainees who train in programs with postdoctoral researchers who have intensive research backgrounds are more likely to apply for and receive subsequent research grant support?. To that end, the goal of this proposal is simple - to provide for the PhD and MD Fellow a combined and integrated immersion experience in a cutting edge cross campus research program that is in itself embedded in and focused upon a more clinical environment where health and disease is the primary consideration, and to then promote the use of nonhuman primate and human derived models in MD or PhD Fellowship projects lead by MD/DVM/PhD Faculty of combined clinical and traditional training backgrounds. The campus home for this program will be the integrated Program in Endocrinology (iPEnd), which was founded in 2016 to foster and promote collaboration focused on fetal development and the compromised adult outcomes of adverse pregnancy. We propose here that iPEnd also offers a vibrant interdisciplinary and multidisciplinary environment comprised of MD, PhD and DVM trained faculty with which both MD and PhD Postdoctoral Fellows could train together to enter the world of translational research. Such a blended training environment is very much needed if we are to maintain a future pool of interdisciplinary translational research team members intellectually and professionally ready to pursue the goals of NICHD to improve reproductive health and outcomes. Another consideration by NIH is that trainees do not go on to future independent success by simple exposure to ?good science? alone. Indeed, both Trainees and Trainers need support and professional education to ensure the best outcomes. To that end, our proposed training includes a deep immersion in higher level research training typical of many T32 programs at this time, but we aim to go far beyond to include the evidence based 8 core competencies for Postdoctoral trainees combined with added Mentee and Mentor training and the complimentary Professional Development Resources in order to achieve the rich blended training environment so strongly recommended by NIH. We believe iPEnd is ready to promote the concepts of the interdisciplinary and multidisciplinary ?Translational Workforce? through the proposed Training Program, operating within first class campus environment geared for basic but increasingly supportive of translational research. On that basis, we submit this application.