Victor M Navarro - US grants

PROJECT SUMMARY This K99/R00 proposal is designed to complement and advance my prior research experience. Briefly, my expertise is in the study of the neuronal mechanisms that modulate reproductive function. I have contributed to the characterization of the roles of kisspeptins (encoded by the Kiss1 gene) as regulatory conduits of gonadotropin-releasing hormone (GnRH) release. During my post-doctoral fellowship, I have broadened this research to include the central modulators of kisspeptin release neurokinin B (NKB) and dynorphin A (Dyn), which are co-expressed with kisspeptin in the arcuate nucleus (ARC). We have proposed a model whereby NKB and Dyn act through recurrent collaterals to shape the pulsatile release of kisspeptin that would ultimately lead to pulses in GnRH release. The overall goal of this research proposal is to understand the physiological function of subpopulations of Kiss1 neurons that regulate the secretion of GnRH and hence reproduction. Kiss1 expressing neurons are found in two discrete nuclei in the hypothalamus, the ARC and anteroventral periventricular nucleus (AVPV), and are direct targets for the negative and positive feedback action of estradiol E2 on GnRH secretion, respectively. Kiss1 expression in these neurons increases at the time of puberty onset and humans and rodents bearing inactivating mutations in this gene or its receptor (Kiss1r) exhibit impuberism and infertility. The objective of this proposal is to use genetic mouse models in combination with viral-dependent gene therapy to dissect the differential role of the ARC and AVPV Kiss1 and NKB neuronal populations in the control of reproduction. We will use genetically engineered mice that express Cre recombinase and GFP under the Kiss1 promoter. Breeding to homozygosity would lead to Kiss1 deficient (but Cre/GFP positive) mice. Using Cre-dependent adeno-associated viral constructs, we aim to reinsert the Kiss1 gene specifically in Kiss1 neurons of the ARC or the AVPV. The specific aims of the project are to: 1) determine the role of Kiss1 neurons in the ARC vs. AVPV in the control of puberty onset in male and female mice; 2) determine the role of Kiss1 neurons in the ARC vs. AVPV in the positive and negative feedback of sex steroids; and 3) determine the role of NKB in the ARC in the control of puberty in male and female mice. The studies outlined in this proposal are designed to advance our fundamental knowledge of reproductive neuroendocrinology?for the development of better strategies to treat reproductive disorders and offer improved methods of contraception. Completion of these studies will provide training in: (a) state-of-the-art genetic and molecular methodologies (b) addition of a translational component to my research studies through close interaction with clinical investigators; and (c) skills necessary for success as an independent principal investigator. This additional training will aid in my development of a competitive research program and contribute to my establishment as an independent investigator.

ABSTRACT The activation of the gonadotropic axis requires the pulsatile release of the hypothalamic decapeptide GnRH. Reproduction is thwarted in the absence of these pulses. Despite this critical feature of GnRH release, the nature of the central mechanisms that govern this pulsatile release remain unknown. In recent years, Kiss1 neurons of the arcuate nucleus have been posed as likely candidates to hold this pulse generator through the coordinated action of its co-transmitters tachykinin neurokinin B (NKB) and dyrorphin A (Dyn), which has led these neurons to be termed KNDy neurons. I propose a model in which NKB stimulates and Dyn inhibits kisspeptin release from KNDy neurons leading to a pulsatile pattern that would then be translated into GnRH, and therefore LH, pulses. However, we have recently documented that other tachykinins (substance P, SP and neurokinin A, NKA) can activate KNDy neurons, adding an additional complex layer to this model. Importantly, recent studies in rodents and monkeys indicate that the action of tachykinins to induce LH release happens at the level of KNDy neurons and is therefore kisspeptin-dependent. However, we have recently observed that in the presence of circulating estradiol (E2) levels, both NKB and SP evoke a potent increase in LH release in a kisspeptin-independent manner, which compromises the previous model. Importantly, KNDy neurons and GnRH neurons (albeit to a lesser extend) express the receptors for SP (Tacr1) and NKB (Tacr3), suggesting that tachykinins may be able to act at both neuronal levels to induce LH release under the right sex steroid conditions. The overall goal of this proposal is to characterize the role of each tachykinin in the frequency and amplitude of LH pulses in the mouse at the KNDy neuron vs GnRH neuron using a series of complementary functional, neuroanatomical and genetic studies. The successful completion of this proposal will offer new strategies to treat reproductive disorders affecting GnRH secretion.

SUMMARY The hypothalamic Kiss1 neurons control gonadotropin-releasing hormone (GnRH) release through the secretion of kisspeptins. Kiss1 neurons serve as a nodal center to convey regulatory cues that determine the attainment and maintenance of reproductive function. Despite this critical role, little is known regarding the mechanisms that ultimately impinge kisspeptin synthesis and release. In our preliminary data using Drop-Seq of arcuate neurons from adult mice, we have identified Nhlh2 as a highly Kiss1 neuron-specific transcription factor (TF) of the basic helix-loop- helix family. Moreover, this TF is inhibited in conditions of negative energy balance, in line with the regulation of the Kiss1 gene. In addition, JASPAR analysis has revealed 12 potential binding sites of Nhlh2 within the 5?UTR of the Kiss1 promoter. Based on these findings, we hypothesize that Nhlh2 may play a crucial role in the development and function of Kiss1 neurons postnatally, as well as in the regulation of kisspeptin synthesis by metabolic factors. To test this hypothesis, we aim to characterize the role of Nhlh2 in Kiss1 neurons through: a) the study of the expression profile of Nhlh2 in the hypothalamus (especially in Kiss1 neurons) across postnatal development; b) ablation of Nhlh2 expression from Kiss1 neurons using Kiss1-cre:Nhlh2fl/fl mice to characterize the reproductive and metabolic role of Nhlh2 in Kiss1 neurons in vivo; and c) confirming the recruitment to the Kiss1 promoter and the enhancer action of Nhlh2 through chromatin- immunoprecipitation (ChIP) assays and in vitro luciferase assays. Altogether, the successful completion of this proposal would significantly contribute to our understanding of the mechanisms that control kisspeptin synthesis, which may lead to the development of new strategies to treat reproductive disorders, such as delayed puberty onset, polycystic ovarian syndrome or hormone- related cancers.

ABSTRACT Successful reproductive function depends on the proper activation of the gonadotropic axis and the development of adequate sexual behavior. While the hypothalamic mechanisms regulating gonadotropin release have been extensively studied, those regulating sexual behavior and specifically those connecting behavior and fertility have remained elusive. The amygdala has been documented to integrate sexual cues, e.g. pheromones, to elicit sexual behavior and lesion studies have indicated its relevance for proper GnRH release in both sexes. Interestingly, the hypothalamic neuropeptide, kisspeptin (Kiss1), critical for reproductive function, has also been identified in the amygdala. Our preliminary data documents that the activation of Kiss1 neurons in the medial amygdala (MeAKiss1 neurons) stimulates LH release. Therefore, our overarching aim is to characterize in detail the role of MeAKiss1 neurons in males and females in the control of gonadotropin release and sexual behavior through a combination of functional, pharmacological and genetic studies that will include: 1) optogenetic and chemogenetic manipulation of cre- expressing neurons in Kiss1-cre mice; 2) ablation of MeAKiss1 neurons using cre-dependent, apoptosis inducing, Casp3 viruses; and 3) track tracing of projections from MeAKiss1 neurons to hypothalamic neurons using fiber markers (mCherry), mono-synaptic track tracers and calcium photometry circuit mapping. Moreover, we will characterize the interaction between these two functions (i.e. gonadotropin release and sexual behavior) by assessing the activation of Kiss1 and GnRH neurons during mating, and the activation of hypothalamic nuclei involved in sexual behavior during the LH surge in females. Overall, the successful completion of this project will significantly extend our understanding of the role of the medial amygdala in the integration of sexual behavior and gonadotropin release, which is fundamental to synchronize mating and ovulation for successful reproduction. The expected findings will offer new platforms for the treatment of reproductive disorders of central origin such as hypogonadism or low sex drive.