Lori Zeltser, Phd - US grants

DESCRIPTION (provided by applicant): The major goal of this application is to elucidate the molecular events that control the expression of the signaling molecule Sonic hedgehog (Shh) in the ventral diencephalon and within the zona limitans intrathalamica (zli). In addition to its well-characterized role in ventral patterning throughout the neural tube, genetic studies suggest that Shh in the zli influences antero-posterior (A/P) growth and patterning of the diencephalon. The ability to manipulate Shh signaling in the ventral diencephalon and zli individually would facilitate the analysis of their role(s) in thalamic development. This application will therefore focus on the mechanisms regulating Shh expression in these two diencephalic domains. First, in ovo and in vitro manipulations of chick forebrain will be used to define tissue sources of signals upstream of Shh expression and to identify candidate molecules. Second, the molecular events that regulate Shh expression will be examined in chick, initially focusing on the roles of two extrinsic inductive signals, BMP2 and Nodal. Third, enhancer elements regulating Shh expression in the ventral diencephalon and zli will be identified in chick explant and transgenic mice. Finally, the role of Shh in zli maturation and diencephalic patterning will be analyzed in conditional knockouts in mice. These studies are intended to identify some of the steps upstream of Shh expression in the diencephalon and thus facilitate the analysis of the individual contributions of signals from the ventral diencephalon and zli to regionalization of the diencephalon. The adult thalamus, the main integration center of the brain, is derived from the diencephalon. Defining the molecular steps that control the development of this structure may therefore provide insight into the formation of neuronal circuitry in the forebrain and the faults in the system that can lead to neurological disease. Furthermore, knowledge of the precise combination of signals required to specify a particular part of the brain may provide insights into the design of new treatment strategies. As a new investigator, these studies will serve as the basis for a traditional research grant application in the future.

DESCRIPTION (provided by applicant): Epidemiological studies have shown that patterns of increased food intake and adiposity in overweight children are predictive of adult obesity, and thus lend urgency to the need for novel approaches to combat the obesity epidemic in children. Research efforts in the past several decades have identified many signals and cellular components of neuronal circuits that regulate food intake and body weight; however, the vast majority of these studies have been performed in mature animals. Mild phenotypes resulting from disruptions of gene function or neuronal ablations from birth highlight the fact that neuronal circuits regulating energy homeostasis have an extraordinary compensatory capacity in young animals. A genetic model of hypothalamic leptin resistance (LeprHYP) provides a system to explore whether these compensatory functions can be harnessed to improve metabolic phenotypes during a critical period of development for circuits regulating energy expenditure and adiposity. LeprHYP mice exhibit early-onset hyperphagia and obesity; however, they maintain stable levels of adiposity from 8 weeks of age. These findings support the idea that baselines for metabolic phenotypes that are established in young LeprHYP mice are defended with maturity. To explore whether altered metabolic parameters in young LeprHYP mice would be defended in adults, LeprHYP mice were pair-fed to the intake of controls from weaning through 10 weeks of age. Adiposity was reduced by ~20% during the pair- feeding, but more importantly, this lower level of adiposity was stably maintained throughout adulthood. These findings raised the possibility that the post-weaning period in rodents represents a critical period of development during which metabolic phenotypes develop in response to their nutrient/hormonal environment. The goal of the proposed studies is to define the temporal (Aim 1), physiological (Aim 2) and spatial (Aim 3) correlates of a putative critical period of development for metabolic phenotypes. The time window of the sensitive period will be more precisely defined by reducing the duration of the pair-feeding (Aim 1, Exp. 1). To examine whether the molecular predicates of the putative critical period are similar to those that operate in sensory circuits, the ability of GABAA receptor agonists to prematurely initiate the onset of the critical period will be assessed (Aim 1, Exp. 2). Analyses in Aim 2 are designed to define the physiological adaptations associated with pair-feeding that persist in adults, as the circuits regulating these phenotypes likely represent an important source of plasticity in the system. Studies in Aim 3 will examine how hypothalamic leptin- sensing circuits interact with other neuronal circuits to regulate metabolic phenotypes. To examine interactions with hypothalamic insulin-sensing circuits, LeprHYP will be crossed to a floxed allele of insulin receptor (Insr) (Aim 3, Exp. 1). The contribution of extra-hypothalamic leptin-sensing neurons to either the reduction in adiposity achieved by pair-feeding and/or its maintenance in adults will be examined in mice with a pan- neuronal disruption of leptin signals (Aim 3, Exp 2).

PROJECT SUMMARY A growing body of evidence supports the idea that brown/beige adipose tissue (BAT) contributes to basal metabolism in adult humans, and that diminished BAT activity can lead to obesity. Sympathetic nerve stimulation increases BAT activity in rodents. Reports that signaling from the stellate ganglion (SG) modulates thermogenesis in humans, raises the possibility that minimally-invasive approaches to stimulate sympathetic projections from the SG to supraclavicular BAT (scBAT) have potential as an anti-obesity therapy. The neuroanatomy of the SG is complex, because sympathetic and sensory neuronal axons leave the ganglion through 6-8 different exit points to reach a wide variety of targets in the periphery. For neuromodulation of SG projections to BAT to have clinical applications, it is essential that stimulation protocols avoid projections from the SG to the heart and upper extremities. In theory, this can be achieved by physically focusing the electrical stimulus to SG neurons as they exit the SG or enter the scBAT depot. Alternatively, genetic approaches could be used restrict pharmacological manipulations to the subpopulation of SG neurons that innervates BAT. These complementary studies in mouse models and human tissue samples will serve as the basis for deciding which of these strategies are viable therapeutic options. The proposed studies are designed to fill critical information gaps and to develop tools needed for comprehensive mapping of neural circuits regulating BAT and to explore the potential use of BAT neuromodulation as an anti- obesity therapy. Studies in Aim 1 will use combinations of transgenic mouse models and fluorescent neuronal tracers to determine whether there is any physical overlap between BAT and forelimb-projecting soma within the SG or their exit points out of the SG. In parallel, we will perform the first mapping studies of the projections from the SG to scBAT in human autopsy specimens. A major obstacle to studies to modulate or record neural activity in the SG is that this ganglion contains many different types of afferent and efferent neurons that regulate a wide range of physiological functions. Studies in Aim 2 will define molecular markers for distinct subpopulations of SG neurons that project to BAT and heart. Then we will determine whether any of these markers are conserved in human surgical samples. Finally, studies in Aim 3 will establish systems to evaluate the impact of neuromodulation on the organization of sympathetic fibers in conjunction with well-established assays to measure BAT oxidative capacity and activity. To aid these efforts, we will develop techniques to image sympathetic projections in an intact BAT depot. In addition, we will validate key in vivo assays needed to assess the impacts of neuromodulation on BAT function that can be readily translated to humans. In addition to impacts on the BAT field, these studies will also provide a strong foundation for future efforts to understand how existing SG neuromodulation therapies have beneficial effects on a wide range of conditions that are refractory to other treatments, including ventricular tachycardia, chronic regional pain syndrome and post-traumatic stress disorder.

PROJECT SUMMARY Anorexia nervosa (AN) has the highest mortality rate of any psychiatric disease, and there are no effective treatments. A major obstacle to identifying new therapeutic targets is the lack of insight into causes of the pathophysiological eating behavior. Malnutrition and co-morbid psychiatric illnesses cause dramatic changes in the brain and periphery that complicate efforts to uncover factors responsible for disease onset. The Zeltser lab developed a new mouse model to study AN at the stage of illness prior to disease conversion by taking advantage of an epidemiological observation that is often overlooked ? genetic susceptibility in adolescence. Female mice carrying an allele associated with genetic susceptibility to AN (BDNF-Val66Met) were exposed to social isolation stress and caloric restriction during adolescence. Approximately 40% of these mice exhibit severe self-imposed dietary restriction, sometimes to the point of death. Studies using this mouse model of the pre-AN state identified a novel therapeutic target for AN treatment: arginine vasopressin receptor 1A (AVPR1A). The proposed experiments will map the AVP? AVPR1A circuits in the brain that are necessary and sufficient to suppress feeding and will determine which are potentiated by gene x environment interactions that promote susceptibility to anorexic behavior. Studies outlined in Aim 1 will utilize pharmacological, genetic and pharmacogenetic approaches to define populations of AVPR1A neurons that are necessary and sufficient to suppress feeding in wild-type mice. In parallel, experiments in Aim 2 will use a combination of retrograde tracing and pharmacogenetic techniques to identify neuronal populations that transmit the anorexic AVP signal. Since there are many distinct circuits that regulate feeding, studies in Aim 3 will determine where anorexic effects of AVP and the expression of AVPR1A pathway components are enhanced in hBDNFMet/? females exposed to peri-pubertal social isolation stress. The elucidation of brain circuits that promote anorexic behavior in our mouse model would provide a strong foundation for future efforts to explore whether AVPR1A antagonists that are currently in Phase II clinical trials for other psychiatric indications could benefit some AN patients. Lessons learned will also significantly advance the understanding of how the common BDNF-Val66Met variant exacerbates the effects of social stress on the adolescent brain to increase susceptibility to a variety of anxiety-related and affective disorders.

The proposed studies will use brown adipose tissue (BAT) as a platform to elucidate how postnatal developmental influences control the neuroanatomical structure of sympathetic nervous system (SNS) inputs and how these changes impart lasting and specific effects on whole body physiology and susceptibility to disease risk. A wide range of developmental exposures influence SNS tone onto metabolically-relevant organs, including pancreas, kidney, adipose tissue and heart. To date, evidence for a relationship between developmental influences on SNS tone and organ function is purely correlational. An obstacle to direct investigations of the contributions of SNS programming to adult physiology is that experimental manipulations of their activity during development would likely have impacts on organ function that affect the overall health of the animal, which would confound interpretation of the results. We identified two early postnatal exposures that specifically reduce the number of sympathetic neurons in the stellate ganglion that project to BAT (SGBAT), but program disparate responses to physiological challenges in healthy adults. Housing at 30°C leads to lasting effects on responsiveness to cold but not high fat diet, while lactation in a small litter (SL) programs susceptibility to diet-induced obesity but not cold. Studies outlined here will establish novel systems to test whether changes in SGBAT number impact innervation and SNS tone onto BAT (Aim 1), and whether they are necessary and sufficient to program lasting effects on organ function and physiological responses to cold and diet challenges (Aim 2). We will leverage our discovery of molecularly distinct subpopulations of SGBAT neurons and differentially expressed genes in BAT from mice raised at 30°C vs. 22°C to uncover molecular mediators of programming in SNS circuits (Aim 3). We will perform parallel transcriptomic analyses in BAT and SG to identify sources of specificity in the functional responses to manipulations of litter size or temperature during a critical period of development. Because transcriptional signatures of SGBAT subclasses are shared by all SG neurons, lessons from these studies can be applied to other postnatal exposures and organ systems that have been implicated in developmental programming of disease risk.

PROJECT SUMMARY-Project #4 The bone-derived hormone osteocalcin (OCN) is released during bone remodeling, a process that declines with age. Physiological functions regulated by OCN, such as fertility, glucose tolerance, and cognition, deteriorate over the lifespan in parallel with circulating levels of the active form of OCN. Here we explore the possibility that OCN regulates another age-dependent process, thermogenesis, through effects on brown adipose tissue (BAT). We consider two types of thermoregulatory dysfunction in aging populations: cold intolerance and hot flashes. BAT is a thermogenic organ that oxidizes fatty acids and glucose to produce heat. Cold or pharmacological stimulation of BAT induces thermogenesis and improves glucose tolerance in mice and humans. Aging is associated with diminished BAT function and increased risks of hypothermia and metabolic dysfunction. Little is known about the causes and consequences of impaired BAT function in aging because most studies in animal models exclusively evaluate young adults. Sympathetic nervous system (SNS) tone, the primary driver of BAT activity, is elevated in aged individuals. Thus, reduced BAT activity in aging reflects hyporesponsiveness to cold and SNS signaling. Factors that can enhance sensitivity to SNS signals or regulate BAT function independent of the SNS have the potential to improve metabolic and thermoregulatory dysfunction that accompanies aging. OCN levels decrease in aging, in parallel with reduced responsiveness of BAT to cold and sympathetic signaling. Conversely, OCN treatment is sufficient to stimulate a thermogenic gene program in brown adipocytes in vitro and to protect against diet-induced obesity (DIO) by increasing energy expenditure and BAT capacity in vivo. Studies in Aim 1 will explore whether loss of endogenous OCN increases susceptibility to cold and DIO via diminished BAT function in Ocn-/- mutants and aged wild-type mice. Studies in Aim 2 will investigate the mechanism by which exogenous OCN protects against DIO and ask whether restoring physiological levels of OCN to aged mice will improve metabolic function and cold tolerance by activating BAT. Studies in Aim 3 will consider another situation where OCN and thermoregulation are dysregulated, peri-menopausal hot flashes. Estrogen depletion in the peri-menopausal period or following ovariectomy is associated with a burst of bone resorption and a transient increase in active OCN levels. This period is often accompanied by thermal dysregulation and vasomotor symptoms (VMS) in the form of hot flashes. Conditions associated with reduced bone absorption and OCN, including treatment with anti-resorptive compounds and obesity, are associated with reduced risk of hot flashes. We will investigate the role of OCN in a mouse model of hot flashes. If successful, this research will provide novel insights into the mechanism underlying sensitivity to cold and DIO in aged individuals and hot flashes in peri-menopausal women understudied issues with widespread effects.