Alexander (Sasha) Kauffman - US grants

PROJECT SUMMARY (See instructions): The Kissi gene encodes proteins called kisspeptins, v^rhich bind to the G-protein coupled receptor GPR54. Kissl is expressed in discrete brain regions that regulate reproductioa and in mammals, induding humans, kisspeptins stimulate the secretion of gonadotropin-releasing hormone (GnRH) and gonadotropins (LH, FSH). Kissl neurons are themselves regulated by gonadal sex hormones, suggesting a crucial role for kisspeptin signaling in the negative and positive feedback regulation of reproduction. Although the stimulatory role of kisspeptin-GPR54 signaling in the control of GnRH secretion has been well-studied, the role of Kissl nemons in the environmental, circadian, and developmental regulation of reproductioivis much less cftaractenzea. ine goals or this research are to elucidate the physiological role Ofthe KiHlil system in the circadian, photoperiodic, and developmental control of GnRH secretion. The first objective is to evaluate the role of Kissl neurons in the drcadian-controlled pre-ovulatory LH surge. In females, the pre-ovulatory LH surge is coupled to a circadian oscillator in the suprachiasmatic nucleus (SCN). Althoiigh the rieural circuitry and neuroendocrine factors that mediate the SCN's control of the surge remain unidentified, evidence implicates the involvement of Kissl neurons. I will investigate whether Kissl neurons undergo circadian activation at the time of daily LH surges and determine whether there are direct neural cormections between the SCN and Kissl neurons (and if so, identify" the neurotransmitters involved in this circuitry). The second main objective is to evaluate the role of Kissl neurons in the seasonal control of reproduction and sexual maturation. It is known that seasoital reproduction and puberty onset are governed by pineal melatonin (MEL) secretion (which reflects day length), but the neural circuitry that integrates and relays MEL signals to GnRH neurons remains unidentified. I wiU assess the effects of photoperiod/MEL on the hypothalamic Kissl system, ascertain whether MEL's effects on Kissl neurons are direct or indirect, and determine how the regulation of Kissl neurons varies with developmental status (puberty). I will also comprehensively evaluate the roles of several candidate nuclei in relaying any indirect effects of MEL on Kissl neurons. Lastly, I will investigate how the irmervatipn of GnRH neurons by Kissl axons and the sensitivity oi Kissl neurons to feedback effects of sex steroids areregulated by photoperiod and developmental status.

DESCRIPTION (provided by applicant): In mammals, including humans, puberty onset reflects the activation of the neuroendocrine reproductive axis, and adolescence is therefore a time of key physiological, anatomical, behavioral, and psychological changes. However, the specific processes timing and governing the activation of the reproductive axis during pubertal maturation and earlier developmental stages remain poorly understood, as does the reason for earlier sexual maturation in girls than boys. Similarly, the reason for a higher incidence of precocious puberty in girls and delayed puberty in boys is unclear. Recently, the neuropeptide kisspeptin, and its receptor Kiss1R, have been implicated in pubertal development and adulthood fertility. Encoded by the Kiss1 gene, kisspeptin stimulates GnRH secretion in mammals, including humans, and mutations in Kiss1 or Kiss1R impair fertility and puberty in rodents and humans. Despite evidence linking hypothalamic Kiss1 neurons to the control of reproduction in adulthood, less attention has recently been given to the role of Kiss1 neurons prior to adulthood. The overall goal of this proposal is to investigate the role of the Kiss1 system in the sex-specific regulation the reproductive axis in postnatal and pubertal development. Aim I will investigate the importance of kisspeptin signaling in the secretion of gonadal steroids during the postnatal "critical period", a process which directs sexual differentiation of the brain. Experiments in this aim will assess whether postnatal gonadal steroid secretion is impaired in mice lacking kisspeptin signaling, if kisspeptin treatment can induce gonadal steroid secretion in postnatal females, and whether Kiss1 neurons in specific brain nuclei are activated during postnatal gonadal steroid secretion. Aim II will explore the role of the Kiss1 system in key stages of pubertal maturation. Experiments in this aim will determine when and where (in the brain) Kiss1 neurons first become activated during peripubertal development, whether changes in Kiss1R comprise a key element of pubertal development, and whether acute, short-term blockade of central or peripheral kisspeptin signaling impairs puberty onset. Aim III will investigate the role of both gonadal hormones and non-gonadal factors in regulating Kiss1 neurons during peripubertal development. Experiments in this aim will analyze whether pubertal changes in hormone sensitivity of the reproductive axis reflect developmental changes in the sensitivity of Kiss1 neurons to hormone feedback, assess the timing of developmental changes in gonadal hormone-independent regulation of Kiss1 neurons in relation to puberty onset, and elucidate whether sex differences in peripubertal Kiss1 neurons are organized by hormones during early postnatal life. Overall, this proposal will provide a better understanding of how and when the reproductive axis is regulated during different critical stages of development, as well as where in the brain such regulation is specifically derived. This information could provide important insight into the mechanisms underlying hypogonadotropic hypogonadism, precious puberty, and delayed puberty. PUBLIC HEALTH RELEVANCE: Adolescence is a time of critical physiological, behavioral, and psychological changes, but the precise molecular, cellular, and neural mechanisms underlying the regulation of pubertal development remain one of the enigmas of modern science. This proposal investigates the role of hormones and kisspeptin signaling in the regulation of the reproductive axis during key periods of development, including the postnatal "critical period" and sexual maturation (puberty). This work will contribute to our understanding of the critical role of hormones and neural circuits in essential reproductive and developmental processes, and will provide further insight into the mechanisms responsible for various human reproductive disorders and diseases, such as idiopathic hypogonadotropic hypogonadism, nutritional infertility, oligomenorrhea, polycystic ovarian syndrome, and precocious or delayed puberty.

Kisspeptin, a recently-identified neuropeptide encoded by the Kiss1 gene, is both necessary and sufficient for puberty onset and adulthood fertility; however, exactly how Kiss1 neurons, which are known to be located in the hypothalamus, are themselves regulated is poorly understood. In females, Kiss1 neurons in the anterior hypothalamus are regulated by temporal signals from the brain?s circadian clock, thereby generating a precisely-timed activation of the reproductive axis that results in ovulation. Interestingly, this circadian regulation of Kiss1 neurons is fully dependent on the presence of the hormone estradiol, but how and where in the brain estradiol acts to induce the circadian activation of Kiss1 neurons is unknown. Additionally, the Kauffman lab has identified a novel extra-hypothalamic population of Kiss1 neurons in the amygdala which is also stimulated by estradiol; however, the molecular mechanisms of this regulation and the functions of amygdala Kiss1 neurons are completely unknown. Using mice, this research will ascertain the neuroanatomical location and molecular mechanism(s) by which hypothalamic Kiss1 neurons are regulated by both hormonal signals and circadian cues, and will also elucidate the hormonal regulatory mechanisms and potential functions of the novel Kiss1 population located in the amygdala. This intellectual merit of this research is a better understanding of how, when, and where in the brain hormones, particularly estradiol, regulate the function of critical pubertal and reproductive neural circuits, in particular, Kiss1 circuits. The research component is complimented and integrated with the outreach and broader impact aspects, which are to directly train and educate undergraduate and graduate students in hands-on biology research, to participate in the UCSD Initiative for Maximizing Student Diversity which engages and mentors undergraduate minority students in biology research, and to design and manage a new dynamic science website (Neuroendo Now) that serves as a public forum for, and disseminates current information pertaining to advances in neuroendocrinology.

Stress can drastically inhibit fertility and reproductive status, but how this happens in the body and brain is poorly understood. Indeed, although the negative effects of stress on reproduction are well appreciated, the specific signaling factors and neural mechanisms that converge in the brain to inhibit reproduction under stressful conditions remain poorly defined. This proposal will use novel mouse models and neural gene profiling to test how psychosocial stress alters inhibitory neural circuits in the brain in relation to controlling fertility. This proposal will substantially expand our knowledge of how stressors "communicate" with specific parts of the brain that control reproduction. This information will significantly advance the field of reproductive neuroendocrinology. Moreover, given the common theme of stress inhibition on reproduction in many animals, this research will ultimately have broad impact for better understanding brain and hormone functioning of multiple vertebrate species. The research component is complemented and integrated with a comprehensive broader impact plan which includes training and mentoring undergraduate students and post-doctoral scholars, including women and under-represented minorities. The proposal also includes a program aimed at engaging under-represented minority high school students in science research, as well as a new brain research outreach program and internship collaboration with a local high school with students from diverse ethnic and socioeconomic backgrounds.

Stress can inhibit fertility and reproductive status, but exactly how this occurs mechanistically is poorly understood. Reproduction is simulated by gonadotropin-releasing hormone (GnRH) secretion from the brain. The highly-conserved neuropeptide RFamide-related peptide-3 (RFRP-3), encoded by the Rfrp gene, negatively regulates the reproductive axis by inhibiting GnRH secretion. However, very little is known about the phenotype, regulation, or functional necessity of RFRP-3 neurons. As an inhibitor of GnRH, RFRP-3 is poised to relay inhibitory stress signals to the reproductive axis, but this requires testing. This proposal will utilize new transgenic RFRP-3 mouse models (the first of their kind), coupled with cutting-edge molecular tools, to functionally test the involvement and necessity of RFRP-3 neurons in mediating the negative effects of stress on reproductive status, as well as identify novel brain genes that are activated under stressful and non-stressful conditions. This study will therefore provide fresh insight into the physiological regulation and functional roles of RFRP-3 neurons and other brain circuitry in the modulation of reproductive status during stress, and will empirically probe RFRP-3 regulation and function in new ways that have not been possible with former histological and pharmacological methods.

PROJECT SUMMARY The brain regulates the reproductive axis via gonadotropin-releasing hormone (GnRH) neurons. GnRH neurons are themselves regulated by ?upstream? neural circuits, but the mechanisms underlying this are still not well known, especially before adulthood. In particular, the processes timing the activation and progression of pubertal development in either sex are poorly understood. How is puberty triggered, and why does it occur when it does? Why do girls enter puberty before boys? These fundamental questions remain unanswered. Recently, the neuropeptide kisspeptin was linked to puberty and fertility. Yet, the precise roles and regulation of the various kisspeptin neural populations in puberty are still poorly understood, as is the timing or necessity of kisspeptin signaling at unique pubertal stages. Moreover, potential sex differences in pubertal kisspeptin timing and action, which may relate to known sex differences in normal puberty and pubertal disorders, are completely unexplored. This proposal uses mouse models to study the functional roles and potential interplays of kisspeptin and inhibitory neural signaling factors, dynorphin and GABA, in puberty control in both sexes. Aim I illuminates the precise temporal and neuroanatomical roles of endogenous kisspeptin signaling before and during key stages of puberty in males and females. Aim I determines 1) whether short-term blockade or enhancement of kisspeptin neuronal firing during key developmental times alters puberty onset or completion and if this differs between the sexes, 2) the necessity of discrete neuroanatomical kisspeptin populations for pubertal onset and progression in each sex, and 3) the molecular profile of peripubertal kisspeptin neurons to ascertain how known reproductive genes and novel identified genes specifically in Kiss1 neuronal populations change their expression with puberty, and whether this differs between sexes. Aim II studies the role of the inhibitory neural factors, dynorphin and GABA, in puberty onset and progression in both sexes. These factors have been implicated in suppressing adult GnRH/LH secretion, but it is unknown to what degree dynorphin or GABA are involved in pubertal timing in either sex or whether they interact directly with kisspeptin neurons to coordinate puberty. Aim II tests 1) if blockade of endogenous dynorphin or GABA signaling advances pubertal onset or completion in males or females, 2) whether endogenous dynorphin or GABA signaling occurring directly in kisspeptin neurons is necessary for normal puberty onset and/or progression in either sex, and 3) whether endogenous dynorphin or GABA signaling comprise a key component of the neural suppression of prepubertal kisspeptin neurons in a sex specific manner to help time puberty onset. Overall, this proposal using cutting-edge transgenic, chemogenic, pharmacologic, and molecular profiling techniques in both sexes to provide new insight into the neural mechanisms?including both stimulatory and inhibitory factors?underlying the triggering and timing of normal puberty and pubertal disorders, which are both sexually dimorphic for reasons not yet known.