SuJean Choi - US grants

The purpose of these experiments is to understand how the ventromedial nuclei (VMN) direct neural and hormonal metabolic signals that may play a role in the development of clinical hypertension, obesity, and diabetes. Recent work from our lab has shown that the regulation of hypothalamic- pituitary-adrenal (HPA) axis function is embedded in a larger hypothalamic system regulates energy balance. The VMN of the hypothalamus are integral to feeding behavior, body weight, adrenocortical activity, and insulin secretion. We propose to study the integrative role VMN have on the HPA axis, feeding, and insulin secretion; we will test the hypothesis that inputs to the VMN from limbic and visceral inputs are involved. First, we will survey VMN's contribution to corticosteroid feedback on HPA function. Second, we will measure the degree to which circadian rhythms in HPA activity, and the effects of fasting on these rhythms, are mediated by VMN- driven input to the hypothalamic paraventricular nuclei (PVN) which contain the neuroendocrine motor output to the HPA axis. Finally, we will try to determine if the mineralocorticoid-receptor directed corticosterone feedback observed in rats with VMN lesions is mediated by inputs to VMN from cell groups in the ventral amygdalo-hippocampal area and whether VMN activity on food intake and HPA axis activity is regulated by cholecystokinin from the lateral parabrachial nuclei.

DESCRIPTION (adapted from the application) The hypothalamic ventromedial nuclei (VMN) are fundamental to the circadian regulation of energy balance. Lesions of the VMN produce hyperinsulinema and a marked reduction in the amplitudes of circadian rhythms of feeding, hypothalamic-pituitary-adrenal function, body temperature, and locomotor activity. The combination of these effects ultimately leads to obesity. Moreover, these circadian rhythms can be shifted from light cues provided by the suprachiasmatic nuclei (SCN; master biological clock) to food cues provided by the VMN. Entrainment of rhythms to food intake occurs when food intake is restricted to a very short period during the light phase (rats are nocturnal and eat during the dark period) and when activity in the VMN is intact. Lesions of the VMN prevent this shift in rhythms. However, the mechanisms and/or circuitry by which the VMN exert these effects on body weight and circadian rhythms are mostly unknown. The hypotheses presented in this proposal are that: 1) the VMN amplify SCN oscillatory outputs that regulate neuronal c-Fos rhythms in brain regions known to control energy balance; and, 2) differentially expressed genes in the VMN during restricted feeding participate in regulation of circadian energy balance by the VMN. Comparisons between sham and SCN lesioned rats will be made to determine if diurnal c-Fos rhythms in the brain are abolished. Identification of differentially expressed genes in the VMN between restrict fed and ad libitum fed rats will be made since VMN are necessary for the entrainment of circadian rhythms by restricted feeding. Suppression subtractive hybridization will be used to isolate and identify differentially expressed genes in the VMN, which may lead to identification of signals used to regulate circadian rhythms and energy balance. Aberrations in circadian regulation of energy balance may be linked to pathologies such as obesity, eating , and affective disorders such as depression. These experiments and techniques contribute to long-term research goals to investigate how the nervous system integrates circadian rhythms that are regulated by several oscillators, the advantages of such a system would provide, and the metabolic consequences of disruption of such a system. Investigating these questions from physiological, endocrinological, and molecular perspectives will help to integrate mechanisms with function. Three years of post-doctoral training at UCSF have already provided strong technical and research skills that have positively shaped the development of a research career for Dr SuJean Choi. The continuation of research at UCSF and in the laboratory of Dr. Mary F. Dallman would provide a multidisciplined approached to research and offer significant advantages for pursuing a research career in an academic setting.

DESCRIPTION (provided by applicant): This pilot and feasibility grant application (NIDDK R21 PA-02-008 announcement) seeks funding to conduct a series of exploratory experiments to characterize the use of RNA interference (RNAi) as a method to suppress the expression of specific neuropeptides in the mammalian brain. RNAi is a recently discovered process that exploits a highly conserved surveillance mechanism for double stranded RNA (dsRNA), to cause a selective, post-transcription gene silencing of homologous mRNA. Thus, exposure of cells to dsRNA coinciding to a specific mRNA should silence the expression of that mRNA and subsequently the peptide. Applied to neurons in brain, RNAi could prove to be a method for the selective elimination of any neurochemically-specific set of neurons that involve a protein in the neuron's signaling mechanism (e.g. neuropeptide transmitters, biosynthetic enzymes). For example, the method could be applied to the suppression of specific appetite modifying peptide neurotransmitters in the paraventricular nucleus of the hypothalamus (PVN) to examine their roles in food intake regulation. The general utility of such an adaptable method to neuroscience would potentially be remarkable. While preliminary data reveal the feasibility of the procedure, it must be evaluated and characterized for its utility and reliability. We propose to evaluate and characterize local application of RNAi by using dsRNA against the neuropeptide galanin (GAL) and the galanin receptor-1 (GAL-R1). We will determine the key parameters (dose, volume, time course of loss and recovery) of using dsRNA against GAL and GAL-R1 selectively in the PVN. We will then use these optimal parameters to evaluate the functional effect of PVN GAL and GAL-R1. Eating behavior will be the functional output examined, because GAL's actions on food intake are well characterized. The results, when completed, will offer a detailed procedure for using RNAi reliably as a highly selective, neurochemically-specific "lesioning" method and evidence of its utility in studying physiological functions of particular neuronal subpopulations in the brain.

DESCRIPTION (provided by applicant): This proposal investigates the influence of serotonin (5-HT) and its associated neurons in the hypothalamic paraventricular nuclei (PVN) on neuronal corticotropin releasing factor (CRF) mRNA expression and feeding behavior in rats. PVN CRF neurons are important mediators in the control of feeding behavior; e.g. exogenous application of CRF or stimulation of CRF neurons result in the inhibition of food intake. Similarly, drugs that stimulate synaptic 5-HT transmission in the brain are also well known to inhibit food intake. Anatomically, it is also known that 5-HT terminal projections are found on PVN CRF neurons; this suggests that 5HT agents may suppress appetite through heretofore uncharacterized actions on PVN CRF neurons. A potential functional link between 5-HT is further supported by data demonstrating the presence of mRNAs for 5-HT receptor subtypes linked to appetite regulation in PVN CRF cell bodies. Remarkably, despite the suggestion of a number of studies, there have been no definitive studies to date demonstrating that 5-HT-associated drugs may indeed suppress feeding by stimulating CRF mRNA production and CRF protein expression. In this proposal, we hypothesize that there exists a functional link between serotonin (5-HT) and neuropeptides, such as CRF and melanocyte stimulating hormone (alpha MSH) and their respective neurons residing within the PVN (5-HT, CRF) and the arcuate (ARC) hypothalamic nuclei (alpha MSH), respectfully. Our Specific Aims are to (1) determine if 5-HT release regulates CRF or alpha MSH neurons in the PVN to suppress food intake; (2) Determine if 5-HT release indirectly regulates CRF neurons in the PVN via 1-MSH neurons residing in the arcuate and its associated neurons; and (3) determine downstream targets of hypothalamic CRF and its associated neurons. In our experimental design, we will utilize fenfluramine (FEN), a drug that strongly promotes 5HT synaptic transmission. Central and systemic FEN administration will be used to assess PVN CRF mRNA expression in rats pre-treated with 5-HT receptor antagonists specific to PVN CRF neurons that are appetite-linked (the 5HT-1B, 2A, & 2C subtypes). FEN will also be used to test whether 5-HT affects PVN CRF mRNA expression indirectly; 1-MSH release onto CRF neurons is stimulatory; hence, FEN could stimulate CRF neurons by increasing 1-MSH signaling. RNA interference will be used to eliminate CRF mRNA, to determine if FEN-initiated 5-HT influences appetite suppression by additional downstream mechanisms. These studies should provide new insight into the role of a poorly characterized 5HT-CRF synaptic connection in the control of food intake, and ultimately contribute to understanding if and how this connectivity is important to the regulation of overall energy balance, and the etiology of obesity, anorexia, and other appetite-related health problems. This proposal investigates the influence of serotonin (5-HT) and its associated neurons in the hypothalamic paraventricular nuclei (PVN) on neuronal corticotropin releasing factor (CRF) mRNA expression and feeding behavior in rats. These studies should provide new insight into the role of a poorly characterized 5HT-CRF synaptic connection in the control of food intake, and ultimately contribute to understanding if and how this connectivity is important to the regulation of overall energy balance, and the etiology of obesity, anorexia, and other appetite-related health problems.

DESCRIPTION (provided by applicant): Abnormal glutamate signaling within corticostriatal pathways has been linked to craving in humans and cocaine seeking in rats. Unfortunately, our limited understanding of glutamate has contributed to the lack of effective, well-tolerated treatments for many CNS diseases, including drug addiction. While glutamate is described as the primary excitatory neurotransmitter in the brain, it is unclear how the many components of this complex network of transporters and release mechanisms function in an integrated manner to regulate excitatory signaling. Due to a lack of available tools that selectively target these novel mechanisms, it has been difficult to convincingly demonstrate the importance of these novel mechanisms. One such component is system xc-, a source of nonvesicular glutamate release that is primarily expressed on astrocytes. It functions by exchanging extracellular cysteine for intracellular glutamate. System xc influences synaptic activity and plasticity through the release of glutamate and dopamine in multiple brain regions. Repeated cocaine produces a persistent reduction in system xc- activity, which appears to be necessary for glutamate-induced compulsive drug seeking. In contrast, manipulations that prevent or reverse cocaine-induced changes in system xc- activity normalize glutamate levels and blunt cocaine-induced reinstatement. In humans, N-acetylcysteine has shown promise in the treatment of drug addiction and related compulsive disorders. Studies such as these indicate that system xc- function may have profound implications in revealing the cellular basis of addiction, as well as the role of astrocytes in central nervous system activity - especially if it is determined that system xc- is the primary mechanism of action for N-acetylcysteine. Efforts to manipulate system xc- in rats typically involve the use of pharmacological tools that are associated with predictable pharmacological concerns. Increasing system xc activity by direct infusion of cystine into the brain or systemic administration of a cysteine prodrug (e.g., N acetylcysteine) are both effective since the rate of cysteine-glutamate exchange is a function of the relative extracellular/intracellular concentration gradients of its substrates. Mutations in the gene giving rise to xCT, the active subunit for system xc, is present in multiple mouse strains. However, essentially every study linking system xc to glutamate homeostasis or addiction has been conducted in rats or primates. The goal of this proposal is to use the novel Zinc Finger Nucleases (ZFN) approach to mutate the Slc7a11 gene encoding xCT in rat. After creating an xCT deficient rat model (aim 1), we will verify and characterize the general phenotype (aim 2) as well as addiction-specific phenotypes (aim 3). The development and application of these technologies to generate transgenic rat strains may result in a major paradigm shift in studying the neural basis of addiction by enabling more sophisticated and highly specific manipulations in a species that better models critical aspects of human addiction.

Project Summary/Abstract The impact of the obesity epidemic in the US has been estimated to endanger the health of up to 30% of Americans by increasing the risk of diabetes, hypertension, stroke, and cancer as well as leading to 300,000 deaths annually. In order to lessen the negative impact of obesity, there is a need to better understand the neurobiology of feeding and metabolism in a manner that could contribute to the development of effective therapeutics. Attempts to understand the neural basis of feeding and metabolism often involve the study of individual neuropeptides that alter caloric intake and/or energy expenditure. Anatomical studies have implicated subregions of the hypothalamus as sites of action for several dozen peptide regulators of feeding behavior and body weight. Specifically, the ventromedial nuclei of the hypothalamus (VMN) exert powerful control over feeding and metabolism through a broad array of both central and peripheral signaling molecules. For example, leptin, a well- studied peripheral signal produces hypophagia when administered into the VMN. Centrally, the actions of the VMN on energy homeostasis are also dependent on the activity of neural inputs synthesized and released by other brain regions such as the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP), which promotes energy expenditure and decreases feeding behavior. Thus, neurons in the VMN likely integrate both neural and peripheral signals in order to regulate energy homeostasis. Preliminary data in this proposal support the hypothesis that leptin and PACAP interact in the VMN by regulating shared intracellular signaling pathways that lead to hypophagia. This is not surprising given the common and parallel features between PACAP and leptin in terms of anatomy, behavioral and metabolic outcomes, gene activation patterns, and functional dependency. This proposal examines several points of convergence among the intracellular signals produced by leptin and PACAP including the JAK-STAT signaling cascade and BDNF expression. Moreover, PACAP and leptin are ideal candidates to study the functional interactions between a tonically peripheral signal and a synaptically released neuropeptide that together could alter the activity of a broad neural network responsible for feeding behavior and energy homeostasis. In the hypothalamic VMN, we will examine the intracellular signaling links between PACAP and leptin (aim 1), and the functional relationship between PACAP and leptin under chronic conditions of leptin receptor dysregulation with obesity, hyperleptinemia without obesity, and diet induced obesity (aim 2).