Holly A. Ingraham - US grants

The process of mammalian sex determination and differentiation is of fundamental importance and remains one of the basic questions of human biology. Recently, strong genetic evidence has determined the piece of the Y-chromosome thought responsible for testes determination. with the discovery of the SRY gene, Sex-determining Region of Y, the beginning of this gene cascade may be defined, and the search for downstream target genes can begin. While the exact identification of downstream targets may prove difficult, experimental evidence suggest that the evolution of the Sertoli cell-type of the testis is intimately linked to the Y- chromosome. As the "indifferent" bipotential genital ridge begins to differentiate into testes, one can visualize the Sertoli cells aligning themselves into cord-like structures. Shortly after, a critical function of the Sertoli cell will be the expression and local release of the hormone, Mullerian inhibiting substance, MIS. During a critical time, the secretion of MIS results in the regression or "cell death" of the Mullerian duct, the anlagen of internal female genitalia. Thus, regulation of MIS gene expression is directly coupled to the differentiation of the Sertoli cell and the execution of the Y- chromosomal developmental programs. Oddly enough, MIS is also expressed at adult granulosa cell of the ovary; functions in this "sister" cell type of the Sertoli cell remain unclear. Here, it is proposed to define and isolate the developmental regulator(s) of MIS gene expression. Preliminary data suggest that a conserved element, MIS-RE-1, close to the promoter of the mouse MIS gene is bound by a Sertoli-cell specific protein. Further, point mutations that abolish binding also decrease transcription activity as measured in transfection assays using primary Sertoli cells. Binding activity of this protein is roughly correlated to the time of peak MIS transcript expression. Moreover, the same MIS- RE-1 element is also bound by an ovarian-specific protein exhibiting distinct binding specificity and a relative molecular weight different from that observed in the testes. Aim #1 will characterize the mouse MIS gene by defining the transcriptional start site and comparing its sequence with that of the human MIS gene. Standard biochemical analyses and functional studies will probe the protein-DNA interactions between the MIS-RE-1 and the testis-specific MIS-REBP (binding protein). Complete ontogeny of testes-MIS-REBP activity and ovary-specific activity will be undertaken. Aim #2 proposes to purify and clone the Sertoli- specific protein binding to the MIS-RE-1 site. The purification and cloning of the testes-MIS-REBP will follow well-established protocols for DNA-binding proteins. If the testes-MIS-REBP acts to initiate MIS-gene expression its transcript and protein expression should correlate with the onset of MIS gene activation. This hypothesis can be tested following isolation of the MIS-REBP cDNA clone. Aim #3 will attempt to characterize the developmental effects of MIS cis-elements by a simple set of transgenic mouse experiments. In choosing to study the initial activation of MIS gene expression, it is likely that these studies will shed light on basic molecular mechanisms underlying the development and maturation of reproductive endocrine organs.

DESCRIPTION (Adapted from the applicant's abstract): The tissue-specific transcription factor, Pit-l, has previously been cloned and shown to regulate two hormones secreted by the pituitary, growth hormone (GH) and prolactin (PRL). Further, Pit-1 transcripts are found in both cell types and appear one day prior to the onset of GH and PRL gene expression. Yet, both of these cell types are able to restrict their gene expression to only one unique hormone. The molecular mechanisms for this additional specificity have not been identified. While the correlation between Pit-1 and GH/PRL gene expression is striking, Pit-1 transcripts and protein are also found in another pituitary cell type, thyrotrophs, known to secrete thyrotropin (TSH). The overall aim of this proposal is to investigate molecular mechanisms responsible for establishing a subset of cell types in the anterior pituitary by defining the combinatorial effectors that interact with Pit-l in normal pituitary development. Several parallel and complementing strategies have been initiated in order to start defining this combinatorial code. The study proposes to identify cellular proteins that interact with Pit-l by a screening method using radiolabeled Pit-1 or its sub-domains to screen an expression library. Two independent yeast screening strategies will attempt to isolate proteins that directly or indirectly interact with Pit-l. Proteins that interact with Pit-l will be characterized as to their patterns of pituitary expression, and the functional consequences of this interaction by in vivo and in vitro analyses.

DESCRIPTION: (Adapted from applicant's description) The long-term research objectives of the investigator are to determine the molecular mechanisms that regulate the expression of Mullerian Inhibiting Substance (MIS) and how MIS signals Mullerian duct regression during mammalian reproductive tract development. With the investigator's recent finding that a major regulator of MIS gene expression in the developing male gonad is the orphan nuclear receptor, steroidogenic factor-l (SF-1) and preliminary identification of a soluble protein factor secreted by the Sertoli cell referred to as Gonadal Activation Factor (GAF) that activates SF-1, the following specific aims are proposed: 1) to complete the characterization of the MIS promoter in vivo; 2) to purify, characterize and clone GAF; and 3) to characterize the mechanism of GAF activation of SF-1. These studies will provide insight into the molecular mechanisms that govern mammalian sex determination and reproductive development and the basis of human sexual disorders.

Mullerian Inhibiting Substance (MIS, AMH) is a distinct member of the transforming growth factor beta (TGFbeta) superfamily. Members of this superfamily are polypeptide growth factors that exhibit diverse effects on normal cell growth, adhesion, mesenchymal-epithelium interactions, cell differentiation and programmed cell death. Mammalian reproductive tract development begins with the selective differentiation of either the Wolffian or Mullerian ducts which give rise to the male or female reproductive tract, respectively. In males, testicular production of MIS triggers destruction of the Mullerian duct, which we hypothesize to involve programmed cell death. The molecular dissection of the MIS signaling pathway provides a unique system to study hormonal induction of programmed cell death between adjacent cellular layers. This type of cell death is postulated to specify much of the morphological remodeling and adjustment of cell numbers during development. We seek to define the molecular death signals emanating from the paramesonephric mesenchyme that initiate apoptosis of the Mullerian epithelium. Presently, only the MIS ligand the MIS Type II receptor have been identified in the MIS signal transduction pathway. Our proposed studies are designed to delineate other components of MIS signaling cascade via the following aims: 1) Identify the Type I MIS receptor. 2) analyze the biochemical signaling events following activation of the Type I & II heteromeric complex. 3) Identify the downstream MIS-induced death signals that result in the involution of the Mullerian duct. These studies will provide a basic biochemical framework for understanding how TGF-beta-like hormones elicit changes in cellular identity, morphology, and cell number using a unique model system. Moreover, given the potential role of MIS and other gonadal TGFbeta-like hormones to act an antiproliferative signals or tumor suppressor genes, our research may also shed light on possible mechanisms that contribute to abnormal cell growth and tumor progression in the reproductive system.

Gordon Research Conferences bring together active researchers in a selected scientific field with the purpose of defining and analyzing important problems, recent advances and opportunities for research through both formal presentations and extensive discussion. The specific aim of this proposal is to obtain partial funding for the 1999 Gordon Research Conference on Hormone Action. The 1999 Conference represents the 30th annual meeting of the Hormone Action Conference, and will be held at Kimball Union Academy in Meriden, New Hampshire. The conference will continue to provide a forum for presentation and discussion of the latest advances in molecular endocrinology and cellular signal transduction. This is one of the few research conferences that covers a broad spectrum of topics related to Hormone Action, including hormones and growth factors that act through plasma membrane receptors as well as hormones that act through intracellular receptors. The 1999 conference will consist of an opening keynote presentation followed by eight Scientific Sessions. Briefly, they will discuss the following: 1) Invertebrate Model Systems and Development of Endocrine Organs, 2) Hormone Control of Signaling and kinase cascades, 3) New Peptides and Receptors in Hormone Action, 4) Hormones and Behavior, 5) Mouse Models in Hormone Action, 6) Orphan Nuclear Receptor Function 7) Chemistry, Structure and Transcriptional coactivators, and finally 8) Integration of Nuclear Receptors and Cofactors in Transcription. Two Poster Sessions are planned for the conference to encourage students, postdoctoral fellows and young investigators to join senior investigators in presenting their work. All meeting participants will be invited to submit an abstract for the Poster Sessions, allowing all attendees the opportunity to present data and fully participate in discussions. Traditionally, this conference brings together a diverse group of academic and industrial scientists with interests ranging from basic researchers studying molecular, cellular and physiological mechanisms of hormone and hormone receptor action, to physician-scientists interested in the integrative actions of hormones and in understanding human endocrine diseases. The limited attendance, focused Scientific Sessions and Symposia, and highly successful Poster Sessions combine to provide an exciting forum for the exchange of ideas and to provide extensive opportunities for participation by a diverse group of scientists interested in Hormone Action.

DESCRIPTION (provided by applicant): Published studies from several labs, including our own, have demonstrated essential roles for the orphan nuclear receptor, Steriodigenic factor 1 (SF-1) in endocrine organogenesis, male sexual differentiation, and steroid homeostasis. Our prior work shows that maximal SF-1 mediated transcription and recruitment of general cofactors depends on a single phospho-serine 203 located in the hinge region, and that receptor phosphorylation mimics several features of ligand-dependent receptor activation. Here, we propose to: Aim 1 Determine the in vivo role of SF-1 serine 203 phosphorylation. Aim 2 Identify cofactors that are recruited upon SF-1 phosphorylation by using a yeast genetic screen, an affinity-GST purification method, or a high-throughput peptide binding assay. Aim 3 Establish the function and dynamics of cofactor recruitment for identified candidates. The potential role for phosphorylation in regulating nuclear receptor activity has been provocative, but remained secondary to ligand-activation of receptors. Now, because some members of the nuclear receptor gene family may not have specific high affinity ligand, post-translational events and protein-protein interactions have assumed a more legitimate position in modulating receptor activity. To date, studies addressing how extracellular signaling and nuclear receptor function are integrated have been limited to cellular model systems. As such, our aim to assess the in vivo function of SF-1 phosphorylation will be of significance. Our preliminary data show that SF-1 is phosphorylated at S203 in endocrine tissue; these observations provide a strong rationale for using mouse genetics to determine the in vivo significance of this modification. We are also excited by our new finding that the UBC-9 SUMOylation conjugating enzyme interacts with SF-1. Indeed, the close proximity of the MAPK phosphorylation site to the perfect SUMOylation consensus site in the hinge region of SF-1 is provocative, and warrants further investigation into the possible interplay between these two posttranslational events. We believe our studies with SF-1 will provide valuable insights into how posttranslational events modulate ligand-independent receptor activation and will be directly relevant to the progression of hormone-insensitive endocrine tissue cancers.

[unreadable] DESCRIPTION (provided by applicant): For several years our laboratory has focused on the role of orphan nuclear receptors in endocrine development and physiology with an emphasis on members of subfamily V that includes steroidogenic factor 1 (SF-1), liver receptor homolog 1 (LRH-1) and the Drosophila Ftz-F1. Broadly speaking, these receptors participate in developmental events as well as in adult functions, including steroid and bile acid homeostasis. However, it remained unclear if subfamily V orphan nuclear receptors are ligand-dependent. We recently obtained the high-resolution crystal structure of the ligand binding domain (LBD) of LRH-1 (Sablin et al., 2003), and find that LRH-1 adopts an active conformation in the absence of ligand. Knowing this, we have pursued how ligand-independent mechanisms, such as post-translational modifications, control receptor activity. In ongoing funded work, we are exploring the structural and biological consequences of receptor phosphorylation, given that SF-1 and LRH-1 are phosphorylated in the C-terminal hinge region. Another posttranslational modification known to alter transcriptional activity is sumoylation or sumo (small ubiquitin-related modifier) conjugation of proteins, similar to ubiquitination - both SF-1 and LRH-1 are sumoylated proteins. Moreover, mutating the key acceptor lysines (K119/K194) in the hinge domain of SF- 1 increases receptor activity implying that sumoylation represses receptor-mediated transcription. These preliminary data prompt three aims: 1) What is needed to achieve full receptor sumoylation and is subnuclear localization and/or promoter occupancy affected by sumoylation? 2) What protein machinery associated with receptor sumoylation mediates repression? And lastly, 3) what are the functional consequences of loss of function sumoylation receptor mutants in Drosophila and in mice? These studies should provide mechanistic insights in SF-1 and LRH-1 activity, and as such, may provide new avenues for modulating orphan receptor activity. Additionally, given that sumoylation of regulatory factors is a new and emerging area in transcriptional regulation, our results will be of interest to the general field of gene regulation. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): For several years our laboratory has focused on the role of orphan nuclear receptors in endocrine development and physiology with an emphasis on members of subfamily V (NR5) that includes steroidogenic factor 1 (SF-1), liver receptor homolog 1 (LRH-1) and the Drosophila Ftz-F1. Broadly speaking, these receptors participate in developmental events as well as in adult functions, including steroid and bile acid homeostasis. However, it has remained unclear if NR5 receptors are ligand-dependent. In a close collaborative effort with Dr. Robert Fletterick's lab at UCSF, we recently obtained the high-resolution crystal structure of the ligand binding domain (LBD) of LRH-1 to 2.4 A resolution, and find that LRH-1 adopts an active conformation in the absence of ligand. Now, we have obtained the crystal structure of mSF-1 LBD to 1.2 A resolution bound by SHP-1 peptide. Comparison of the SF-1 and LRH-1 structures reveals overall similarities, as well as some striking differences. By contrast to the empty pocket observed for mLRH-1, the mSF-1 pocket is occupied by phospholipid phosphotidyl glycerol (PG). Moreover, the region in mSF-1 spanning helices H2 and H3 is much more disordered and flexible than that found in mLRH-1. Here, we propose to extend our structural analyses of NR5 receptors. In our first aim we will assess how differences in helices H2 and H3 affect receptor function, and obtain the crystal structure of coregulatory peptides bound to SF-1/LRH-1, as well as Drosophila, Ftz-F1. Our second aim will address the potential relevance of phospholipids as bona fide ligands for NR5 receptors using biochemical and physiological assays. Finally, in our third aim, we will focus on defining structural changes that occur following posttranslational modification of these receptors, including sumoylation and phosphorylation. We believe that the structural and biochemical studies proposed here will complement greatly the emerging biological framework for this important and biomedical relevant subfamily of nuclear receptors.

DESCRIPTION (provided by applicant): Modulating Nuclear Receptor Activity via Sumoylation Steroidogenic factor-1 (SF-1) is a constitutively active nuclear receptor that regulates critical aspects of adrenal function and the hypothalamic-pituitary-gonadal axis. SUMO modification of nuclear receptors including SF-1 and other NR5A receptors is proposed to repress their transcriptional activity. We examined the functional and structural consequences of SF-1 sumoylation at two conserved lysines Lys119 and Lys194 that reside adjacent to the DBD and LBD, respectively. Surprisingly, while previous loss-of-function studies in cells predicted that sumoylation at Lys194 would greatly impact SF-1 function, the conformation and coregulator recruitment of fully sumoylated SF-1 LBD protein were identical to non-sumoylated protein;only a decrease in Ser203 phosphorylation was noted. By contrast, a selective loss of DNA binding at non-canonical SF-1 targets was observed using DBD protein sumoylated at Lys119. Moreover, regulating SF-1 function by Lys119 sumoylation must occur prior to DNA binding. We propose that sumoylation of transcription factors is an active posttranslational mark that eliminates recognition of "SUMO-sensitive" target genes and restrains gene expression. While these molecular and biochemical studies suggest that sumoylation restrains the transcriptional output of SF-1, this hypothesis needs to be tested in vivo. To this end, a SUMO-deficient mutant knock-in mouse was created in the SF-1 allele, referred to as SF-12KR/2KR. This mutant mouse exhibits embryonic lethality at mid-gestation (E9.5-13.5), which is surprising considering that SF-1 null mice die postnatally due to adrenal insufficiency. Here we will test the hypothesis that normal transcriptional programs are inappropriately regulated in a sumo-deficient SF-1 mutant using whole animal, genomic, and structural approaches. This competitive renewal will address three remaining questions as to the role of SF-1 sumoylation: 1) What developmental or hormonal pathways are subverted in SF-12KR/2KR mice that lead to mid-gestation lethality. 2) What specific elements and genes are inappropriately regulated by the 2KR SF-1 mutation in the placenta and other endocrine tissues? 3) What are the structural consequences of SF-1 sumoylation on the DNA binding domain. Answers to these questions should provide in vivo and structural mechanistic insights as to how sumoylation modulates gene expression. PUBLIC HEALTH RELEVANCE: Post-translational modifications regulate the activity of many types of cellular proteins. Sumoylation of proteins involves protein conjugation and in general dampens the activity of many transcriptional regulators. In this study we will assess for the first time, how a sumo-deficient mutation of the nuclear receptor affects endocrine tissue development and function. Results from this work will potentially identify important new gene targets that are especially sensitive to the relative pools of sumoylated protein. They will also provide mechanistic insight into how sumoylation restrains and "fine tunes" transcriptional programs of the endocrine system.

DESCRIPTION (provided by applicant): Molecular Mechanisms of Atrazine in Altering the Endocrine Gene Network Atrazine (ATR) is widely used as a broadleaf herbicide in the United States and world. While toxic levels of ATR are linked to reproductive abnormalities in wild life, effects in humans remain controversial. Moreover, the molecular and cellular targets of ATR are still unknown. We and others have found that ATR stimulates the nuclear receptor steroidogenic factor 1 (SF-1, NR5A1). In the last decade our lab has worked extensively to define the molecular mechanisms regulating members of subfamily V (NR5A) receptors that includes SF-1, LRH-1, Ftz-f1, ff1a-d. As such, we have begun to ask how ATR functions in mammalian endocrine cell lines and in zebrafish to affect NR5A receptor activity. Our preliminary data show that environmentally relevant ATR concentrations increased the zebrafish (z)Cyp19A1 expression encoding aromatase, and increased the ratio of female to male fish. ZCyp19A1 is a known target of the nuclear receptor SF-1 (NR5A1). By contrast, the second zebrafish gene encoding aromatase zCyp19A2 was unchanged after ATR treatment. Remarkably, in mammalian cells ATR functions in a cell-specific manner to upregulate SF-1 targets and other genes critical for steroid synthesis and reproduction, including Cyp19A1, StAR, Cyp11A1, hCG, FSTL3, LHss, INH1, 1GSU, and 11ss-HSD2. Further cellular studies suggest that ATR increases the activity of mammalian and zebrafish NR5A receptor orthologs by receptor phosphorylation, and amplification of cAMP and investigator3K signaling. Importantly, our data show that ATR does not directly affect SF-1 DNA-binding, nor does ATR appear to directly bind SF-1. Instead, we hypothesize that this environmental herbicide potentially influences endocrine gene networks by indirectly activating NR5A receptors as well as other cAMP-mediated transcriptional events. The precise molecular targets of ATR that result in cell-specific stimulation of NR5A receptors remain to be defined. In this narrowly focused R21 application, two aims are planned to identify the molecular targets of ATR. In our first aim, a systematic genome siRNA screen will be carried out on ATR-responsive reporter cell lines. In our second aim, we will test different categories of candidate targets of ATR. Identifying the underlying mechanism(s) of ATR stimulation of NR5A receptors will begin to address the potentially adverse endocrine disrupting effects of this pervasive and persistent environmental chemical. PUBLIC HEALTH RELEVANCE: Endocrine disrupting chemicals (EDCs) such as the herbicide atrazine (ATR) are widely used and are proposed to adversely affect the endocrine system. Although the consequences of EDCs on the reproductive health and viability of some wild life are established, the precise effects on human health remain controversial. In the United States, use of ATR is heavy in many agriculturally settings, especially in the Midwest where corn is grown, however this herbicide is banned in most EU countries. In this application we seek to define the precise molecular targets of ATR using mammalian culture cells. Our proposed experimental plan to systematically define the molecular targets of ATR is an important step in addressing the potential impact of ATR on wild life and human health and the controversy surrounding its continued use in the US. These studies will also shed new insight into the activation of NR5A receptors as important regulators in reproductive development and physiology.

DESCRIPTION (provided by applicant): Developmental Programs of Neural Circuits in the Mouse Medial Hypothalamus Neural circuits in the hypothalamus control energy balance by adapting and responding to peripheral signals. However, in humans the high incidence of obesity and insulin resistance associated with Type 2 Diabetes illustrates how metabolic needs are easily uncoupled from appropriate behavioral responses. Identifying factors that modulate the development of these neural circuits may provide novel tools for understanding and controlling human obesity. Using a gene discovery approach we identified ~200 molecular markers that begin to define neuronal subtypes in the neonatal ventromedial hypothalamus (VMH). We hypothesize that eliminating these factors in mice will alter cell fate and compromise normal function of the VMH, some of which are linked to energy homeostasis. To begin testing this hypothesis we will use, or will create mouse model systems. Once produced, we will correlate changes in molecular profiles with phenotypic changes in these mutant mice. We posit that sorting out or classifying VMH neurons according to their molecular signatures will provide the necessary tools to be able to rationally carry out further functional analysis (I.e. electrophysiology). The identification of several novel factors expressed in the neonatal VMH provides an exciting, but significant challenge - namely, how does one choose which of these to study in detail given the cost and time of mouse genetic experiments. In this application we focused on a handful of factors because first, we have obtained floxed mouse lines or the funds to create lines, and second, published data from our lab and others suggests that these factors will be important for VMH development. Our broad approach will be to create different mouse models using a combination of floxed alleles that can be crossed to Cre-Recombinase driver lines. In three aims we will ask how Nkx2.1 and Vgll2 function to specify VMH cell fates, and we will determine how elimination of three SF-1 target genes, Nkx2.2, FezF1 and A2bp1, affects VMH development and function. We will use standard immunohistochemistry, in situ hybridization, and microarray profiling analyses to determine how gene expression and cellular patterns change in different mouse mutants. We will also use SF-1 reporter mice that we have generated;SF-1 positive neurons are labeled with the transneuronal marker, WGA and the Tau-GFP reporter. This approach will be complemented when possible with cellular studies using immortalized hypothalamic cell lines. Limited metabolic and behavioral analysis will be carried out to determine if there are obvious physiological consequences in different mouse mutant lines. In this ambitious and somewhat exploratory application our goal is to begin defining how VMH neuronal subtypes develop and how they are potentially linked to physiology. PUBLIC HEALTH RELEVANCE: Childhood obesity has become one of the major public health concerns for the 21st century and as such, the incidence of Type 2 diabetes is now prevalent in young children. Major advances in the study of body weight regulation have identified central nervous system circuits involving the hypothalamus that controls energy metabolism. Despite this knowledge, the increasing rise in obesity underscores how easily food intake is uncoupled from energy expenditure in our society. It is reasonable to hypothesize that defects disrupting the development of normal neural circuits could predispose an individual to obesity. To begin understanding how these circuits are established a gene discovery approach was undertaken and several new proteins were identified that potentially control hypothalamic development and function. In this application we will begin assessing the role of these new brain markers in the development of neuroendocrine circuits. This application is part of an emerging research area that asks whether early developmental events before or after birth influence the risk of metabolic diseases later in life. Accordingly, results from the proposed studies may provide a new arsenal to combat the growing and costly effects of obesity and Type 2 diabetes.

DESCRIPTION (provided by applicant): Food intake and physical activity are coordinately regulated and phased with the female reproductive cycle. While estrogen receptor alpha (ER? in the hypothalamic arcuate nucleus (ARC) modulates food intake, hormone-responsive neurons influencing female locomotion remain undefined. Here, we identify a sexually dimorphic neuronal cluster in the ventromedial hypothalamus (VMH) solely dedicated to promoting physical activity in females. In a loss-of-function study we find that deleting the developmental transcription factor Nkx2-1 with Sf1Cre ablates a subpopulation of ventrolateral VMH neurons expressing ER?and results in marked obesity and reduced locomotion only in these Nkx2- 1Sf1Cre mutant females. Conversely, using a gain-of-function approach we show that directly activating intact Nkx2-1 ventrolateral VMH neurons elicits a female-specific and hormone-dependent burst of physical activity. Loss of gonadal hormone severely blunts this response. Our findings demonstrate that specialized Nkx2-1+ VMH neurons constitute part of a previously undefined sexually dimorphic locomotor circuit that is used specifically in females to combat sedentary behavior and maintain metabolic fitness. Here, we plan to define and understand how this hormone-responsive neural circuit promotes physical activity in females. In three aims, we will 1) assess responses in a different settings to activating DREADDs, 2) determine if estrogen and other potential neuromodulators within VMHvl neurons affect DREADD-induced activity and 3) begin identifying VMHvl markers and mapping projections that are missing/altered in the Nkx2-1Sf1Cre mutant females. Nkx2-1Sf1Cre female mice offer an excellent opportunity to understand the neural basis of gender and hormonal regulation of energy expenditure. Our ultimate goal is to translate this basic research into the development of pharmacological and/or transplantation therapies that can be used to restore metabolic health in females.

DESCRIPTION (provided by applicant): This proposal seeks to continue the successful IRACDA Scholars in Science (ISIS) Postdoctoral Program between the University of California San Francisco (UCSF) as the research-extensive campus and San Francisco State University (SFSU) as the partner institution with a large population of students who are underrepresented (UR) in the sciences. The primary goals of the ISIS Program are: 1) To prepare UR postdoctoral scholars to obtain, and succeed in, competitive faculty positions at excellent academic institutions; and 2) To enable ISIS Scholars to positively impact SFSU by helping develop a research-oriented science curriculum, serving as role models to SFSU undergraduate science students, and promoting research collaborations between UCSF and SFSU faculty. The ISIS Program prepares postdoctoral scholars for academic research and teaching careers through a series of focused activities that develop high-quality research, teaching, and professional skills. To accomplish these goals, the ISIS Program has four specific aims: 1) Provide an extensive mentored-research experience at UCSF so that ISIS postdocs conduct cutting-edge biomedical and behavioral research and publish in high quality journals; 2) Develop an intensive mentored-teaching experience at SFSU, while contributing notably to a research-oriented science curriculum at SFSU. The ISIS Scholars will refine their teaching skills, serve as role models for undergraduates at SFSU, and engage in research-related instruction; 3) Provide career-development opportunities such as learning to write successful grants, and learning to manage a research laboratory in an inclusive and ethical manner, in order to enhance their skills and preparation for an academic career; and 4) Broaden the Impact of the ISIS Program on SFSU Students and Research Infrastructure. Relevance of this program to public health: In order for our Nation to improve its technical and scientific competitiveness, efforts must be made to increase the number of participating scientists and engineers from under-represented groups. This requires a long educational pathway. The IRACDA Scholars in Science Postdoctoral Program addresses two parts of that pathway; inspiring undergraduates to enter a scientific research career, and preparing postdoctoral scholars for faculty careers where they will be able to address the Nation's biomedical, behavioral and clinical research priorities. Developing this potential will ultimately improve our national competitiveness and prosperity.

Project Summary/Abstract: Pilot and Feasibility Program The P&F Program will be a major pillar on which the success of the UCSF-NORC will be built. This program will provide $10,000-$50,000 awards (for a total of $150,000 per year) for proposal that are meant to be exploratory in nature. P&F Awards will allow applicants to collect the preliminary data needed for subsequent grant applications necessary to develop sustained, long-term new research directions. The intentions of this program relate directly to the major UCSF-NORC goal of strengthening a growing interactive community of Obesity & Nutrition researchers by: · Facilitating and allowing new investigators and junior faculty to establish NORC-relevant research programs. · Attracting scientists with established research programs in other areas into the fields of nutritional sciences, obesity, and related disorders. · Fostering highly innovative directions in basic and clinical obesity- and nutrition-related research, including clinical studies in minority and other underserved patient groups. · Developing new and innovative collaborations among NORC investigators and between NORC investigators and other faculty. A straight forward guideline driven review process will be implemented to select P&F proposal for funding. The request for applications will be conducted through a UCSF-wide website, the Research Allocation Program, that today lists 27 intramural funding opportunities. UCSF investigators routinely turn to this site to identify intramural research opportunities, which ensures that the NORC P&F program reaches its primary target of investigators (those new to the nutrition, obesity and metabolism field). The review process will involve extra- mural review of applications following an initial assessment by an ad hoc internal NORC committee including the UCSF-NORC P&F Director and the Directors of the NORC Programs. That same committee shall allocate grants based upon the recommendations of the external reviewers while ensuring a mixture of funded projects in a diversity of areas. The committee also will identify established researchers who, when required, can mentor P&F Awardees who are junior and/or new to the field. Collection of success metrics, regular review of the program by the executive committee and advice from the external board will ensure the continuation of a high quality UCSF NORC P&F program.

PROJECT SUMMARY ? UCSF IRACDA Scholars Program The need for increased representation of a diverse workforce in biomedical research at the academic level is clear and well-documented. This renewal application requests resources to continue our four-year Scholars training program at the University of California, San Francisco (UCSF) in partnership with San Francisco State University (SFSU). The goal of the program, which began in 2007, is to increase the number of highly trained, successfully funded and sustainable research scientists, who will perform high level laboratory and teaching and increase the diversity of younger trainees in biomedical research careers to ultimately improve our understanding of health-related science. To this end, UCSF has partnered with SFSU to build a program that provides comprehensive science and teaching training for mentoring Ph.D. scholars who are committed to an academic career and impacting underserved minority trainees. The key elements of our training program are (1) a mentored research experience, (2) a mentored teaching experience at SFSU, (3) monthly career development workshops, (4) interdisciplinary scientific experiences and (5) continuing review and evaluation. The centerpiece of the program is the 4-years of training, with the initial three years covered by the training grant and the last year covered by the PI funds or Independent Development Awards. The majority of this training (75%) is devoted to intense research training in basic laboratory science with the remaining training devoted to teaching experiences at the SFSU campus and mentoring experiences with undergraduate underrepresented students from SFSU. Our exceptionally well- qualified faculty and teaching mentors have committed their support to our IRACDA scholars. Internal and external Advisors as well as periodic formal evaluations will be a critical part of continued excellence and achievement of stated goals of the Program. Each Scholar is expected to interact with a team of at least three Faculty Research Mentors and two Teaching Mentors during their training; meeting on a regular basis to assist him/her in setting, and then monitoring training and career goals. Our program has a strong track history of recruiting exceptional Scholars representing a diverse background. Of the 35 current and past scholars, nearly 90% are URMs. Of the 21 Scholars who have completed their training at UCSF, they have produced an average of 3.4 peer- review publications (0-9 range), and over 50% have assumed academic positions, with 80% of these at R1/R3 Research Institutions (Vanderbilt, UCSD, UCSF, U of Arkansas, Duke, Georgetown, SFSU and SJSU). We believe the Program's strong outcomes data, the collaborative research-intensive training environment at UCSF, and the well-established and fruitful interactions between UCSF and SFSU provide compelling rationale for renewing and expanding this unique training opportunity to train and diversify the next generation of leaders in biomedical science.

Project Summary/Abstract Our current understanding of mechanisms underlying visceral pain, including that associated with irritable bowel syndrome, remains rudimentary. Importantly, opiates are ineffective at treating visceral pain syndromes, and only exacerbate discomfort by producing constipation, reflecting a clear need for alternative treatment options. The goal of this proposal is to bring greater mechanistic insight to this underserved area of pain research, and to approach the problem in a multifaceted strategy designed to maximize the relevance of our basic research discoveries to future pain treatments. Here, we will ask how enterochromaffin (EC) cells transmit noxious signals from the gut lumen to the spinal cord. EC cells are key sensory cells in the intestinal epithelium that release serotonin onto primary sensory nerve fibers, thereby evoking a sensation of discomfort and pain in response to luminal irritants, such as bacterial metabolites, inflammatory agents, or ingested chemicals. The goals of this collaborative effort are to use activating and silencing approaches to examine functional connections between EC cells and sensory nerve fibers. We will couple these methods with transcriptome profiling, viral tracing, and electrophysiological methods to gain insights into the molecular and functional identity of these fibers. Another key goal is to determine whether EC cell signaling pathways exhibit sex-specific differences, an important question that may relate to the higher prevalence of GI visceral pain syndromes experienced by women. Our team brings an unusually wide ranging and innovative approach to this area of pain research that includes expertise in the neurophysiology, pharmacology, and anatomy of nociceptive and pain circuits, visceral tissue anatomy and development, and relevant clinical experience. This knowledge base is supported by complementary technological approaches that will enable us to connect molecular and mechanistic insights to physiology, visceral nociception, and disease. Our focus on the epithelial-nociceptor connectome highlights EC and other enteroendocrine cell types as potentially powerful control points for neuromodulation of visceral discomfort and pain. A comprehensive functional, pharmacological, genetic and anatomical characterization of EC-primary afferent-spinal circuits is an essential first step toward achieving this important goal. As such, our research program fits squarely within the SPARC mandate to transform our understanding of peripheral nerve-organ interactions and advance strategies for controlling organ system function.

Project Summary/Abstract The goal of this revised R01 is to determine how a hypothalamic region integrates and then processes hormone and CNS metabolic cues to affect sex-dependent physical activity behavior in female mice. We showed previously that loss of hormone-responsive neurons in the medial basal hypothalamus (MBH) diminishes activity only in female mice. In new work, we discovered that a small, discrete cluster of melanocortin 4 receptor (Mc4r)-expressing neurons in the ventrolateral region of the ventromedial hypothalamus (VMHvlMC4R neurons) increases female activity and promotes weight loss when activated. Increasing dosage of Mc4r in the VMHvl of wild type female mice by CRISPRa increases distance traveled leading to weight loss when pair-fed with control mice. Here, we will ask if this VMHvlMC4R node can be exploited to mitigate age-related and environmental- induced metabolic challenges and also define the mechanistic pathway and circuits responsible for regulating this module. Aim 1 will ask if the VMHvlMC4R node depends on hormones, can improve metabolic deficits, and whether silencing this node (inhibitory DREADDs) results in lowered physical activity behaviors. Aim 2 will confirm that Mc4r is an integral part of this node and define other signaling components that participate in the VMHvlMC4R activity node. Aim 3 will address the physiological consequences of melanocortin and estrogen signaling in modulating VMHvlMC4R neuron activity by electrophysiology brain slice recordings with the Gao lab (Yale) and begin mapping inputs that interact with VMHvlMC4R neurons. We posit that this ancillary VMHvlMC4R female-module disengages in states of low or no estrogen (estrus or post-menopause period) thereby reducing metabolic fitness. Our approaches to define the molecular basis of this VMHvl activity node are highly innovative and likely to provide novel insights into how fluctuating hormone-status in a normal cycling female or during different life-stages drives physical activity behaviors.

Project Summary/Abstract In this revised R01 application we will ask how estrogen signaling in the central nervous system controls bone metabolism in a sex dependent manner. It is well known that peripheral estrogen affects bone in both mice and humans. As part of our efforts to understand how estrogen neural circuits in mice affect female metabolism, we discovered that estrogen signaling in the arcuate nucleus (ARC) of the hypothalamus normally suppresses cortical and trabecular bone mass in female mice. Ablating estrogen receptor alpha (ERa) in three intersectional and independent mouse models leads to a striking high bone mass phenotype in female mice; male bones are unaffected. Mutant females exhibit exceptionally dense trabecular and cortical bones, whose strengths surpass other reported mouse models. Stereotaxic- guided deletion of brain ERa confirmed the central origin of this bone phenotype and uncoupled this phenotype from changes in circulating estradiol, testosterone, or leptin. Our work defines central regulation of bone metabolism, alongside reproduction and energy balance, as a fundamental sex- difference in physiology. These data also offer proof for the concept that the brain is a major determinant of female bone metabolism, thus expanding the gatekeeper function of the hypothalamus in energy expenditure to non-classical metabolic tissues, such as bone. Although our research is in the earliest pre-clinical stages, understanding how our models build massively dense and strong bones could transform current rational design of therapeutics for hormonal and age-related bone loss. Why and how female ARC neurons normally inhibit bone metabolism remain a mystery. In the following three aims, we seek to unravel this puzzle. We will define and then confirm which ARC neurons mediate this brain-bone connection, ask if manipulating these neurons via chemogenetics and pharmacology mimics high bone phenotype or normalizes the bone phenotype in mutants, and then begin determining the molecular basis for this sex-dependent high bone phenotype.