Hormonal Analyses in Individuals with Prader-Willi Syndrome and Others with Early-Onset Morbid Obesity

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Hormonal Analyses in Individuals with Prader-Willi Syndrome and Others with Early-Onset Morbid Obesity
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Kweh, Frederick A
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Doctorate ( Ph.D.)
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University of Florida
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Medical Sciences, Genetics (IDP)
Committee Chair:
Driscoll, Daniel J
Committee Members:
Wallace, Margaret R
Resnick, James L
Atkinson, Mark A

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ghrelin -- hyperphagia
Genetics (IDP) -- Dissertations, Academic -- UF
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Abstract:
Prader-Willisyndrome (PWS) is a rare genetic disorder characterized by infantile hypotoniaand failure-to-thrive, developmental and intellectual disabilities,hypogonadism, short stature, behavioral problems, and early-onset morbidobesity.  PWS is caused by failure ofexpression of paternally inherited genes in chromosomal region 15q11.2-q13.  In PWS obesity is the major cause ofmorbidity and mortality; it typically begins at age 1-4 years and is furthercompounded by the development of hyperphagia, resulting from a lack of sense ofsatiety.  The mechanism(s) behinddevelopment of obesity and hyperphagia in PWS remain unclear. Here I report thatghrelin, a potent appetite-stimulating hormone, is significantly elevated ininfants and young children with PWS before the onset of obesity andhyperphagia.  Ghrelin levels were thehighest in PWS infants still in the poor appetite phase.  Given this fact, it is unlikely that elevatedghrelin levels are causing the switch to the hyperphagic phases of PWS.  However, it has been shown in mice thatghrelin can also act to increase fat mass independent of its effect on appetite(Perez-Tilve et al, 2011).  Therefore, itis likely that the elevated ghrelin levels are causing the increased fat massseen in PWS infants compared to normal infants with similar body mass indices(BMI). I also report herethat peripheral leptin levels are elevated in obese PWS individuals whilealpha-melanocyte stimulating hormone (alpha-MSH) and brain-derived neurotrophicfactor (BDNF) are not elevated.  Giventhe fact that BDNF is primarily produced in the brain, and hypothalamic leptinsignals through alpha-MSH to promote BDNF expression, it is therefore possible thatobese individuals with PWS have low brain leptin levels resulting frominadequate brain leptin uptake, and thus suffer from leptin resistance.
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by Frederick A Kweh.
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Thesis (Ph.D.)--University of Florida, 2012.
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Adviser: Driscoll, Daniel J.
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1 HORMONAL ANALYSE S IN INDIVIDUALS WITH PRADER WILLI SYNDROME AND OTHERS WITH EARLY ONSET MORBID OBESITY By FREDERICK A. KWEH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Frederick A. Kweh

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3 To my grandparents whose love and support I have never been without

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4 ACKNOWLEDGMENTS I would like to thank my graduate advisor Dr. Daniel J. Driscoll and my supervis ory committee, Dr. Jim Resnick, Dr. Mark Atkinson and especially Dr. Margaret Wallace to whom I owe so much I thank Clive Wasserfall for providing me with initial guidance with ELISA and Multiplex assays and Dr. Mark Atkinson for allowing me to use his laboratory equipment. I also thank Carlos Sulsona for his immense help collecting, organizing, assaying and analyzing patient samples. I would also like to thank Dr. Jennifer Miller and the entire Driscoll Lab, past and present members, for their help in making this project a possibility. Last, but no t the least, I would li ke to thank Mercedes Rivera for all her support and encouragement

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 ABSTRACT ................................ ................................ ................................ ..................... 9 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 11 Prader Willi Syndrome ................................ ................................ ............................ 11 The Nutritional Phases of Prader Willi Syndrome ................................ ................... 12 Development of Obesity in Prader Willi Syndrome ................................ ................. 13 Ghrelin: The Hunger Gene ................................ ................................ ...................... 13 The Hypothal amic Leptin Melanocortin Signaling Pathway ................................ .... 15 Brain Derived Neurotrophic Factor ................................ ................................ ......... 18 Significance of This Work ................................ ................................ ....................... 21 2 METH ODOLOGY ................................ ................................ ................................ ... 26 Study Subjects ................................ ................................ ................................ ........ 26 Blood Sample Collection and Processing ................................ ............................... 26 Hormone Assays ................................ ................................ ................................ .... 27 Statistical Analysis ................................ ................................ ................................ .. 27 3 ANALYSIS OF GHRELIN LEVELS IN YOUNG AND OLD INDIVIDUALS WITH PRADER WILLI SYNDROME ................................ ................................ ................. 29 Ghrelin Level in Young PWS Children Is Unclear and Controversial ...................... 29 Ghrelin Is Elevated in Infants and Young Children with PWS (0 1.99 Years) ................................ ................................ ................................ ........... 2 9 Ghrelin Is Not Eleva ted in PWS Children 2 4.99 Years Old ........................... 30 Ghrelin Is Not Elevated in PWS Children 5 11.99 Years Old ......................... 30 Ghrelin Is Elevated in Teenagers and Young Adults with PWS (12 20.99 Years) ................................ ................................ ................................ ........... 31 Hyperghrelinemia Precedes Obesity and Hyperphagia in PWS ....................... 31 Correlation of Ghrelin with Leptin, BMI z S core, DEXA, and Growth Hormone T herapy in PWS ................................ ................................ ............ 31 4 ANALYSIS OF LE PTIN SIGNALING IN IN DIVIDUALS WITH PRADE R WILLI SYNDROME THROUGH ANA LYSIS OF CIRCULATING ALPHA MELANOCYTE STIMULATI NG HORMONE LEVELS ................................ ............ 44

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6 Hypothalamic Leptin Signaling Is Unknown in PWS ................................ ............... 44 Leptin Is Appropriately Elevated in Obese Hyperphagic PWS Children ........... 45 Alpha MSH Is Not Elevated in Obese Hyperphagic PWS Children .................. 46 5 ANALYSIS OF CIRCULAT ING BRAIN DERIVED NEUROTROPHIC FACTOR LEVELS IN INDIVIDUAL S WITH PRADER WILLI SYNDROME ............................ 50 Brain Derived Neurotrophic Factor and Prader Willi Syndrome .............................. 50 Serum BDNF Is Elevated in PWS Subjects ................................ ...................... 50 BDNF Levels Decrease with Onset of Hyperphagic Nutritional Phases in PWS ................................ ................................ ................................ .............. 51 6 DISCUSION ................................ ................................ ................................ ............ 56 Hyperghrelinemia Begins Early in Prader Willi Syndrome ................................ ...... 56 PWS Individuals May Suffer From Leptin Resistance ................................ ............. 57 7 FUTURE DIRECTIONS ................................ ................................ .......................... 60 Analysis of POMC and PRCP Expression in PWS ................................ ................. 60 Analysis of Ghrelin Responsive Pathways in Young Children with PWS ................ 60 LIST OF REFERENCES ................................ ................................ ............................... 61 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 67

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7 LIST OF TABLES Table page 1 1 Nutritional Phases in Prader Willi syndrome ................................ ...................... 25 3 1 List of subjects, observations and characteristics ................................ ............... 33 3 2 Results of biological assays and measurements ................................ ................ 34 3 3 Mean change in Dependent Variable (DV) per Unit Change in Independent Variable (IV): Age 0 1.99: Slope (SE)[N]{P value} ................................ .............. 35 3 4 Mean change in Dependent Variable (DV) per Unit Change in Independent Variable (IV): Age 2+: Slope (SE)[N] {P value} ................................ ................... 36 4 1 Leptin and alpha MSH levels in PWS, Sib.C & EMO subjects ........................... 47 5 1 Subjects, observations and biological values for BDNF analysis ........................ 52

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8 LIST OF FIGURES Figure page 1 1 PWS chromosomal region 15q11.2 q13. ................................ ............................ 22 1 2 Ghrelin induction of appetite. ................................ ................................ .............. 23 1 3 The Hypothalamic Leptin melanocortin signaling Pathway. ............................... 24 3 1 Box plot of ghrelin levels in infants and young PWS and normal control (Sib.C) children 0 1.99 years old. ................................ ................................ ....... 37 3 2 Box plot of ghrelin levels in PWS, Sib.C and EMO children 2 4.99 years old. .... 38 3 3 Box plot of ghrelin levels in PWS, Sib.C and EMO children 5 11.99 years old ... 39 3 4 Ghrelin is elevated in teenagers and adults with PWS. ................................ ...... 40 3 5 Bar chart of average ghrelin levels in PWS nutritional phases ........................... 41 3 6 Growth hormone therapy decreases ghrelin levels in PWS ................................ 42 3 7 PWS children have low weight for length and normal body fat .......................... 43 4 1 Box plot of peripheral leptin levels. ................................ ................................ ..... 48 4 2 Box plot of peripheral alpha MSH levels ................................ ............................. 49 5 1 Graphical representation of subject ages and BMI z scores .............................. 53 5 2 Box plot of serum BDNF levels ................................ ................................ ........... 54 5 3 BDNF levels decrease with onset of hyperphagia in PWS ................................ 55 6 1 Working model for development of obesity and hyperphagia in PWS ................ 59

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9 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy HORMONAL ANALYS E S IN INDIVIDUALS WITH PRADER WILLI SYNDROME AND OTHERS WITH EARLY ONSET MORBID OBESITY By Frederick A. Kweh August 2012 Chair: Daniel J. Driscoll Major: Medical Sciences Genetics Prader Willi syndrome (PWS) is a rare genetic disorder characterized by infantile hypotonia and f ailure to thrive developmental and intellectual disabilities, hypogonadism, short stature, behavioral problems, and early o nset morbid obesity. PWS is caused by failure of expression of paternally inherited gene s in chromosomal region 15q11.2 q13. In PWS obesity is the major caus e of morbidity and mortality; it typically be gins at age 1 4 years and is further compounded by the development of hyperphagia, resulting from a lack of sense of satiety. T he mechanism( s) behind development of obesity and hyperphagia in PWS remain unclear. Here I report that ghrelin, a potent appetite stimulating hormone, is significantly elevated in infants and young children with PWS before the onset of obesity and hyperphagia. Ghreli n levels were the highest in PWS infants still in the poor appetite phase. Given this fact, it is unlikely that elevated ghrelin levels are causing the switch to the hyperphagic phases of PWS. However, it has been shown in mice that ghrelin can also act to increase fat mass independent of its eff ect on appetite (Perez Tilve et al 2011). Therefore, it is likely that the elevated ghrelin levels are causing the increased

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10 fat mass seen in PWS infants compared to normal infants with similar body mass indice s (BMI) I also report here that peripheral leptin levels are elevated in obese PWS individuals while alpha melanocyte stimulating hormone (alpha MSH) and brain derived neurotrophic factor (BDNF) are not elevated. Given the fact that BDNF is primarily pro duced in the brain, and hypothalamic leptin signals through alpha MSH to promote BDNF expression, it is therefore possible that obese individuals with PWS have low brain leptin levels resulting from inadequate brain leptin uptake, and thus suffer from leptin resistance

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11 CHAPTER 1 INTRODUCTION Prader Willi S yndrome Prader W illi Syndrome (PWS) is a genomic imprinting obesity disorder caused by lack of expression of paternally inherited genes in the PWS region o f chromosome 15q11.2 q13 [Figure 1 1] PWS was first described by Andrea Prader, Heinrich Willi and Alexis Labhart in 1956; it was the first disease to be linked with the phenomenon of genomic imprinting and also the first identified disorder resulting from maternal uniparental disomy (Prader Willi Syndrome A ssociation, USA; Bittel and Butler, 2005; Cassidy and Driscoll, 2009) PWS occurs in 1 in every 20,000 births and equally affects males and females of all races and et hnic groups. The clinical features of PWS include infantile lethargy and hypotonia causing poor feed and failure to thrive, developmental and intellectual disability, hypogonadism, morbid obesity if uncontrolled, hyperphagia, behavioral and psychiatric di sturbances, short statue, temperature and pain insensitivity, characteristic facial appearance and bo dy habitus (Bittel and Butler, 2005; Cassidy and Driscoll, 2009) The l oss of paternally expressed genes in PWS occurs via three major genetic mechanisms: a 5 7 Mb deletion of the paternally inherited chromosome 15q11.2 q13 region, maternal uniparental disomy (UPD) 15, or a defect in the imprinting process in the 15q11.2 q13 region on the paternally inherite d region. Howeve r most cases of PWS result from the sporadic 5 7 Mb deletion with one of two proximal break points (BP1 and BP2) and a distal break point (BP3) [Figure 1 1] I n families where the father carries imprinting deletions in the PWS region the risk is much hig her (Bittel and Butler, 2005; Cassidy and Driscoll, 2009)

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12 The Nutritional Phases of Prader Willi Syndrome Individuals with PWS are typically grouped in to two classic nutritional phases: before and after onset of obesity and hyperphagia. Most recently, children and adults with PWS have been grouped in to four major nutritional phases with sub phases occurring in the first two (Miller et al., 2011) [Table 1 1] Nutritional phase 1 occurs from birth to infancy and usually the affected infant is hypotonic and not obese. Infants in sub phase 1a are characterized by poor appet ite and failure to thrive while infants in sub Phase 1b appear to grow stead ily along at a normal rate and with a normal appetite. Nutritional phase 2 generally occurs between 18 36 months of age and is characterized by significant weight increase across growth percentile lines. Affected children in sub phase 2a have significant weight increase, crossing 1 2 or more growth percentile lines without significant increase in calories or appetite. In sub phase 2b the affected chi ld is typically overweight an d daily caloric intake has increased along with an abnormally increased interest in food. However, the child can still feel full at this stage after a meal. Nutritional phase 3 involves the development of hyperphagia, accompanied by aggressive food seek ing and lack of satiety. The onset of phase 3 is quite variable and may appear as early as 3 years of age or as late as 15 years or, in a small minority, never. Most individuals in this phase are typically obese. Nutritional phase 4 occurs when an indivi dual previously in phase 3 no longer has insatiable appetite and can feel full. This phase does not start until adult hood and even though patients may still have a greater than normal appetite, it is not as aggressive and unrelenting as in phase 3 (Miller et al., 2011)

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13 Developm ent of O b esity in Prader Willi Syndrome In PWS, obesity is the major cause of morbidity and mortality. It typically begins between 1 4 years of age, if food intake is not strictly controlled, and is further compounded by the development of h yperphagia later on in childhood (Cassidy and Driscoll, 2009; Miller et al., 2011) Th e etiology of obesity in PWS r emains unclear: the mechanism(s) behind failure to thrive and poor appetite early on and the subsequent onset of obesity well before any significant increase in appetite and food consumption remain unknown So far, no obesity associated gene has been identified in the PWS region (15q11.2 q13) Ghrelin and leptin have been reported by many groups to be sig nificantly elevated in obese adults with PWS however their role s in PWS obesity remain unclear (Cummings et al., 2002; Delparigi et al., 2002; Goldstone, 2005) A recent study of a small group of PWS patients reporte d low levels of brain derived neur otropic factor (BDNF), an anorexigenic neuropeptide that signals downstream of the hypothalamic leptin melanocortin pathway to regulate food intake and energy expenditure (Han et al., 2010) Here we investigate the onset and consequences of hyperghrelinemia in individuals with PWS We also investigate hypothalamic leptin signaling in obese PWS individuals by assaying for serum concentrations of the melanocortin peptide alpha melanocyte stimulating hormone ( MSH) and the neuropeptide BDNF Ghrelin: The Hunger Gene Ghrelin is a pleiotropic hormone that is secreted at different developmental stages by a wide variety of organs including the pancreas, duodenum, stomach, hypothalamus, pituitary, bone, ovary, testis, and cartilage (Steculorum and Bouret, 2011) It is the first

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14 identified hunger hormone and it was discovered in the rat and human stomach shortly after the discove ry of its receptor ( Kojima et al., 1999) Originally, ghrelin was described as a 28 amino acid peptide that was secreted primarily from the stomach and that bound the growth hormone secretagogue receptor 1a (GHS R1a) to stimulate the release of gro wth hormone (Kojima et al., 1999) Subseq uent studies however revealed ghrelin to be a pleiotropic hormone secreted by a variety of organs with strong appetite inducing effects (Steculorum and Bouret, 2011) is are potentially long lasting; it modulates develo pment of appetite related brain centers early on in life by counteracting the effects of leptin, an anorexigenic hormone secreted by adipocytes which play an important regulatory role in the development of hypothalamic neurons that regulate feeding and ene rgy homeostasis (Bouret et al., 2004a; 2004b; Steculorum and Bouret, 2011) Ghrelin is significantly elevated in adults with PWS (Cummings et al., 2002; Delparigi et al., 2002) however the consequence of this hyperghrelinae mia is unclear; sustained reduction of ghrelin levels with pharmacological agents in PWS subjects did not reverse hyperphagia or significantly alter body composition (Tan et al., 2004; De Waele et al., 2008) Thus the high ghrelin levels observed in adult PWS subjects do not appear to be the cause of their hyperphagia. T he impact of hyperghrelinaemia in PWS however, may occur at an earlier stage suc h as the perinatal period, impacting development of appetite regulatory neurons in the hypothalamus and resulting in long lasting effects Currently ghrelin level s in infants and young children with PWS, as reported in the literature, are not well

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15 descr ibed and appear controversial. Cumm ings et al. reported that ghrelin was not elevated in young non obese children with PWS while Haqq et al. reported that only a subset of young non obese P WS children had hyperghrelinaemia (Erdie Lalena et al., 2006; Haqq et al., 2008) However Tauber et al. reported that hyperghrelinae mia was present in PWS children at any age, and preceded the onset of obesity (Feigerlov et al., 2008) I have analyzed serum ghrelin levels in infants, young children and adults with PWS and at the different nutritional phases of PWS I report here that ghrelin is elevated early on in infants with PWS before the on set of obesity and hyperphagia (Chapter 3) The Hypothalamic Leptin Melanocortin Signaling Pathway Dysfunction of the hypothalamus in PWS is thought to play an important role in the devel opment of obesity and hyperphagia in individuals with PWS. Since the PWS region (15q11.2 q13) does not contain any known obesity associated genes, dysfunction of the hypothalamic leptin melano cort in signaling pathway (Figure 1 3 ) and its downstream effect or, brain derived neurotrophic factor (BDNF), bears explor ing as a cause of the obesity. Leptin is an anorexigenic hormone that is critical in the regulation appetite, energy homeostasis and body weight. sis and body weight is mediated in the hypothalamus, the site of highest mRNA expression of the long isoform of the leptin receptor (Ob Rb), through the hypothalamic melanocortin signaling pathway (Oswal and Yeo, 2007; Farooqi and O'rahilly, 2008) Leptin diffuses into the arcuate nucleus (ARC) of the hypotha lamus and acts dire ctly as a transcription factor i n two distinct classes of pr imary leptin responsive neurons: one

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16 class co expresses the anorexigenic peptides pro opiomelanocortin (POMC) and Cocaine and Amphetamine Related Transcript (CART) which inhibit appetite, while the other co expresses the melanocortin antagonists neuropeptide Y (NPY) and agouti related protein (AgRP) which induce appetite (Oswal and Yeo, 2007) Leptin, upon binding the leptin receptor, triggers a signaling cascade leading to transcriptional activation of the POMC and CART genes, while inhibiting the release of NPY and AgRP (Oswal and Yeo, 2007; Farooqi and O'rahilly, 2008) The full length POMC protein is cleaved by prohormone convertase 1 (PC1) and prohoromone convertase 2 (PC2) in a tissue lipotrophin, and the melanocortin peptides adrenoc orticotrophic hormone (ACTH), alpha beta and gamma melanocyte stimulating hormone s ( MSH). The melanocortins mediate their effect through a family of five G protein coupled receptors known as the melanocortin receptors (Oswal and Yeo, 2007; Farooqi and O'rahilly, 2008) The melanocortin receptors (MCRs) signal primarily through the cyclic AMP transduction pathway via a Gs protein and a denyl cyclase The agouti related protein ( AgRP ), which is inhibited by leptin, competes with alpha MSH for the receptors and acts as an antagonist and an inverse agonist to the receptors (Ollmann et al., 1997; Yang et al., 1999; Nijenhuis et al., 2001) A ctivation of the melanocortin 3 (MC3R) and the melanocortin 4 (MC4R) receptors also stimulate s extracellular signal regulated kinases (ERK) activation, suggesting multiple signaling pathways may be involved in addition to the cAMP pathway (Farooqi and O'rahilly, 2008) Of the five melanocortin receptors, only MC3R and MC4R have been linked with regulation of energy homeostasis Mutations in the MC4R gene result in childhood

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17 obesity, and are the most common monogenic cause of human obesity (Adan et al., 2006; Butler, 2006; Shimizu et al., 2007; Lee, 2009; Roth et al., 2009) P rimary leptin responsive neurons in the hypothalamus make numerous connections with second order hypothalamic nuclei in the lateral hypothalamus (LH), the paraventricular nucleus (PVN), the ventromedial nucleus (VN), and the dorsomedial nucleus (DMN), all of which highly ex press MC3R and MC4R (Oswal and Yeo, 2007) Transcriptional activation of POMC by leptin leads to stimulation of MC4R receptors by alpha MSH in the hypothalamus, resulting in decrease d appet ite and increa sed energy expenditure [F igure 2 1] The functional role of the leptin melanocortin signaling pathway in the development of obesity and hyperphagia in individuals with PWS remains unclear. A decrease in circulating leptin levels, such as during fasting, is accompanied by simultan eous reduction in transcription of POMC and CART genes, and marked increase in NPY and AgRP mRNA levels. P eripheral l eptin is appropriately elevated with the degree of adiposity in individuals with PWS (Butler et al., 1998; Proto et al., 2007) and e xpression of both NPY and AgRP genes appear to be normal in obese individuals with PWS (Goldstone et al., 2002) L eptin induction of POMC gene transcription also appears unhindered in mouse models of PWS (Ge et a l., 2002; Bittel et al., 2007) Thus the immediate mediators of leptin signaling appear t o be preserved in PWS. However, hypothalamic leptin levels and anorexigenic leptin signaling through alpha MSH and MC4R in the brain remain s unclear and unexp lored in PWS. The development of morbid obesity early on in PWS is reminiscent of young children with MC4R dysfunction. Mutations in the MC4R gene causing intracellular

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18 retention of the receptor associate with early age of onset and greater severity of obesity (Lubrano Berthelier et al., 2003) E xpression of MC4R is not known to be a berrant in individuals with PWS however dysfunction through internalization and desensi tization of the receptor due to continuous stimulation by elevated levels of alpha MSH remains a possibility. R ecent studies in mouse hypothalamic GT 7 cells (Shinyama et al., 2003) HEK 293 cells (Gao et a l., 2003) and N2A cells (Mohammad et al., 2007) demonstrate that continuous stimulation of MC4R with high concentrations of alpha MSH causes intracellular retention and desensitization of the receptor. While POMC has been shown to be elevated in the hypothalamus of newborns of PWS mouse models (Ge et al., 2002; Bittel et al., 2007) and peripheral leptin is known to be significantly e levated in individuals with PWS, not much is known about POMC expression or leptin levels in the hypothalamus of individuals with PWS. Thus it is possible that leptin activation of POMC mRNA expression in obese PWS patients is dysfunctional. In this disse rtation, I examined serum alpha MSH levels in non obese and obese individuals with PWS along with lean normal controls and non PWS individuals with EMO. I report here that serum alpha MSH levels decrease with onset of obesity and hyperphagia in PWS subjec ts (Chapter 4). Brain Derived Neurotrophic Factor Brain derived neurotrophic factor ( BDNF ) is a neuropeptide that is widely expressed in the central nervous system (CNS) and is best known for its role in regulating brain development and plasticity (Hofer and Barde, 1988; Carter et al., 2002; Chan et al., 2006) BDNF plays a critical role in neuronal survival and differentiat ion during development of the CNS and in regulating synaptic activity, neurotransmission

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19 and plasticity in mature neurons (Farias, 1999; Cohen and Greenberg, 2008) Its levels in the brain are dramat ically increased during postnatal development with the highest levels of mRNA and protein in the hippocampus, amygala, cerebral cortex and hypothalamus (Bartkowska et al., 2010; Ji et al., 2010) The human BDNF gene is localized on chromosome 11p14.1 and spans over ~70 kb. It contains 11 exons (I V, Vh, VI VIII, VIIh and IX) and 9 functional promoters that are used in a tissue specific and brain region specific manner (Jones and Reichardt, 1990; Pruunsild et al., 2007) transcripts, producing three precursor pro BDNF protein isoforms (a,b,c) that differ in length of their s ignal peptide. Pro BDNF binds preferentially the p75 neurotrophin receptor (p75 NTR ), a member of the tumor necrosis fact or superfamily. Pro BDNF is proteolytically cleaved extracellular ly by plasmin and m atrix metalloproteinase, or in the golgi network b y furin and proconvertase to give rise to mature BDNF protein, a 120 amino acid peptide that binds preferentially to the tyrosine kinase receptor B (TrkB) receptor, promoting development and differentiation of neurons, cell survival, long term potentiation and synaptic plasticity (Hofer and Barde, 1988; Carter et al., 2002; Chan et al., 2006) Current research in animals has implicated BDNF and its receptor TrkB in modulation of energy balance downstream of the melanocortin pathway (Nakagawa et al., 2000; Xu et al., 2003 ; Unger et al., 2007) M ice lacking either one copy of the Bdnf gene or with a tissue specific conditional deletion of Bdnf in the post natal brain developed obesity and hyperphagia (Rios et al., 2001) Similarly, mice with a hypomorphic mutation in TrkB, resulting in 25% normal leve ls of expression, also

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20 develop obesity (Xu et al., 2003) Bdnf mRNA expression in the ventromedial hypothalamus (VMH), an important satiety center in the brain, was shown to be increase d in mice in the fed versus the fasted state. Central administration of BDNF reduced obesity in obese lepr (db/db) mice (Nakagawa et al., 2000) and reversed hyperphagia and obesity in heterozygous Bdnf knockout mice and in Mc4r deficient mice (Xu et al., 2003) suggesting BDNF plays a r ole in mediating energy balance downstream of the leptin melanocortin pathway Nicholson et al showed that activation of MC4R with an agonist in mice leads to the release of BDNF in the brain and regulation of appetite, body temperature and cardiovascular function (Nicholson et al., 2007) Heterozygous Bdnf knockout mice also displayed diminished pain sensitivity and behavioral disturbances (MacQueen et al., 2001) In humans, the clinical phenotypes associated with BDNF deficiency are similar to those observed in individuals with PWS. BDNF deficiency, as observed in WAGR (Wilms tumor, aniridia, genitourinary anomalies, me n tal retardation) syndrome patients with hetero zygous deletion of BDNF (Han et al., 2008) and in an 8 year old girl with disruption of BDNF expression caused by interstitial 11p inversion (Gray et al., 2006) is associated with severe hyperph agia, childhood obes ity, developmental delay s, and decreased circulating BDNF levels A de novo TrkB mutation was detected in an 8 year old boy with severe hyperphagia, obesity, and impaired learning and memory (Yeo et al., 2004) Extremely obese but otherwise healthy children were reported in a 2006 study to have decreased serum BDNF concentration (Areeg H El Gharbawy, 2006) suggesting inappropriately low B DNF may associate with the pathophysiology of morbid obesity.

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21 Most recently, a study of 13 PWS patients reported lower serum and plasma BDNF concentrations, suggesting insufficient central BDNF production in individuals with PWS (Han et al., 2010) In this dissertation, I examine d a larger cohort of PWS pa tients and their normal sibling counterparts along with non PWS individuals with early onset morbid obesity to d etermine their serum BDNF levels I report here that BDNF levels decrease with onset of obesity and hyperphagia in PWS subjects (Chapter 5) Significance of T his W ork Morbid obesity and hyperphagia are the major medical concern s for individuals with Prader W illi syndrome. The mechanism(s) behind their development in PWS remains unclear. Hyperghrelinaemia is present in adult PWS patients but its role or onset remains unclear and g hrelin levels in PWS children remain controversial. Peripheral l eptin is significantly elevated in obese PWS individuals but its hypothalamic levels and signaling remain unknown in PWS Here, I show that ghrelin is significantly elevated in infants and young children with PWS before the onset of obesity and hyperphagia and t hat this may contribute to the increased a diposity observed in PWS infants and the subsequent weight increase and onset of obesity early on in childhood I also show that obese PWS individuals may suffer from leptin resistance despite elevated perip heral leptin levels, as their serum alpha MSH and BDNF decre ase with onset of obesity suggesting low brain leptin levels I present a model of a potential mechanism by which obes ity and hyperphagia develops in hyperghrelinaemic PWS subjects with elevated peripheral leptin

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22 Figure 1 1. PWS c hromosomal region 15q11.2 q13 with breakpoints (BP1, BP2, BP3), genes and their imprinting st atus indicated N on i mprinted genes are in green and imprinted genes are in blue. Angelman syndrome (AS) genes are in orange

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23 Figure 1 2 Ghrelin induction of appetite. Ghrelin secreted in the stomach inhibits the vagus nerve, thereby relieving repression of hypothalamic ghrelin neurons. This results in local release of ghrelin in the hypothalamus, stimulation of NPY/AgRP/GABA neurons, induction of appetite, inhibition of the POMC/CART neurons, and a decrease in energy expenditure

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24 Figure 1 3. The hypothalamic l eptin melanocortin signaling p athway. Leptin secreted by the adipose tissue diffuses into the hypothalamus, binds the leptin receptor (Ob Rb) and trigger POMC transcription. POMC prohormone is cleaved by pr ohormone co nvatase (PC1 & PC2) to release alpha MSH that activates MC4R, leading to decreased appetite and increased energy expenditure.

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25 Table 1 1. Nutritional Phases in Prader Willi syndrome Nutritional Phase Description 0 Dec reased fetal movements, birth weight 15% < normal sibs 1a Hyp o t onia with difficulty feeding & decreased appetite (0 9 mo) 1b Improved feeding, appetite & growth (9 25 mo) 2a Weight increases without increase in appetite or calories (2.1 4.5 yr) 2b Increased appetite, but can feel full (4.5 8 yr) 3 Insatiable appetite, rarely feels full (8 yrs adulthood) 4 Appetite no longer insatiable & can feel full (some adults)

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26 CHAPTER 2 METHODOLOGY Study Subjects Subjects with Prader Willi syndrome before and after growth hormone therapy were recruited to the University of Florida Natural History Study (2001 2011) and admitted in our 2 day intensive research study at the General Clinical Research Center (GCRC) a t Shands Hospital at the University of Florida. Non PWS subjects with early onset morbid obesity (EMO) as well as normal weight sibling controls (Sib.C) were also recruited to serve as comparison group for the study. A medical geneticist and an endocrino logist examined each research subject, and a nutritionist also performed a thorough nutritional assessment. Each subject had blood taken after an overnight fast for collection of plasma and serum, some of which was stored in multiple aliquots at 80 o C unt il further assay A total of 60 PWS subjects, 39 EMO subjects and 95 normal sibling controls from 0 36 years of age were recruited in this study (Table 2 1). Most subjects had blood drawn more than once at different time points during the course of the s tudy, thus accounting for the discrepancy between subject number and sample number. Blood Sample Collection a nd Processing Blood was taken from each research subje ct between 8 9 AM in the morning after an overnight fast. For isolation of serum samples, blood was collected in a vacutainer tube lacking anti coagulants and allowed to sit at room temperature for 15 30 minutes until clot. The samples were then centrif uged at 3000 rpm (1,800 x g) for 10 minutes at +4 o C Both s erum and plasma samples were aliquot into 1mL cryo tubes and stored at 80 o C until use.

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27 Hormone Assays For ghrelin analysis, s erum samp les were analyzed in triplicate using a fluorescent Enzyme linked Immunosorbent Assay (ELISA) kit from Phoenix Pharmaceuticals, Inc., enzyme immunoassay. Briefly, the immunoplate is pre coated with a secondary antibody and the nonsp ecific binding sites blocked. Primary ghrelin antibody and unknown serum samples are incubated overnight in the immunoplate, followed by incubation with a biotinylated ghrelin peptide. The Fc fragment of the primary antibody binds the secondary antibody in the immunoplate, while its Fab fragment competitively binds ghrelin peptide in the unknown samples or the biotinylated ghrelin peptide. The biotinylated peptide interacts with streptavidin horseradish peroxidase (SA HRP) that catalyzes the substrate. The fluorescence intensity is inversely proportional to the amount of ghrelin peptide in the unknown sample. The ghrelin concentration in the unknown sample is determined by extrapolation from a standard curve of known concentrations. For leptin analysis, plasma samples were diluted 2x and assayed with the Luminex assay system using the metabolic panel per instructions of the manufacturer (Millipore Inc, CA, USA). Statistical Analysis ELISA data was normalized for inter assay variability to four internal c ontrols that were present in every assay that was performed. Two tailed test was used to statistically compare groups after adjusting for age and sex differences P values

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28 less than 0.05 were considered to be significant. ion analysis was used to further analyze interactions in groups.

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29 CHAPTER 3 ANALYSIS OF GHRELIN LEVELS IN YOUNG AND OLD INDIVIDUALS WITH PRADER WILLI SYNDROME Ghrelin Level in Young PWS Children Is Unclear and Controversial The consequences of hyperghrelinemia in adult individuals with PWS remain unclear. T he current literature on ghrelin in young PWS children is also unclear and controversial. Studies from the laboratories of Merlin Butler and David Cummings describe ghrelin as normal in chil dren with PWS (Butler et al., 2004; Erdie Lalena et al., 2006) Cummings et al. looked at both acylated and total ghrelin in PWS children and did not find any significant difference from normal controls. Andrea Haqq later reported ele vated ghrelin levels in a subset (33%) of young PWS children (Haqq et al., 2008) However another study by Maithe Taub er reported that hyperghrelinaemia was pre sent in PWS children at any age and preceded the onset of obesity (Feigerlov et al., 2008) In this study, I investigate ghrelin levels in infants and yo ung children with PWS and how it relates to obesity and hyperphagia in PWS Ghrelin I s E levated i n Infants and Young Children with PWS (0 1.99 Y ears) A competitive ghrelin ELISA assay was used to analyze serum ghrelin levels of PWS infants and young children, and normal control infants and young children between the ages of 0 1.99 years [Table 3 1] Ghrelin was significantly elevated in PWS infants and young children relative to normal control infants and young children of the sa me age (5521 3696 pg/ml vs 2883 1172 pg/ml; p=0.016 ) [Figure 3 1 ; Table 3 2 ] There was no significant difference in plasma leptin levels between PWS and normal control infants and young children of this age (272 231 pg/ml vs 216 145 pg/ml; p=0.48 )

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30 PWS infants and youn g children at this age (0 1.99y) had significantly low er weight for length percentile relative to their normal counterparts of the same age. However, body fat percentile as determined by DEXA did not differ significantly between the two groups at this age ; PWS infants had as much body fat as normal sibling control infants [Figure 3 7] despite their low weight for length percentile Ghrelin Is Not Elevated in PWS Children 2 4.99 Y ears O ld Ghrelin levels of PWS children 2 4.99 years old did not di ffer significantly from levels in normal lean control children (Sib.C) and EMO children of the same age ( p=0.12 & p= 0.71 respectively) Also, g hrelin levels i n EMO children 2 4.99 years old did not differ significantly from levels in normal lean children ( p=0.30 ) of the same age [Figure 3 2]. However, PWS and EMO children of this age had significantly higher plasma leptin levels than normal lean control children of this age ( p<0.001 ) [Table 3 2 ] The BMI Z score of PWS children was n ot significantly higher than that of Sib.C but was significantly less than that of EMO children [Table 3 1]. Ghrelin I s Not Elevated in PWS Children 5 11.99 Years O ld Ghrelin was not significantly elevated in PWS children 5 11.99 years old re lative to no rmal lean control children of the same age ( p=0.21 ); however their ghrelin was significantly elevated relative to that of EMO children of same age ( p=0.012 ) [Figure 3 3] Plasma leptin was significantly elevated in both PWS and EMO children within this age group relative to normal lean controls ( p<0.001** ) [Table 3 2 ]. There was no significant difference between leptin levels in PWS and EMO subjects in this age group ( p=0.56 ).

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31 Ghrelin Is Elevated in Teenagers and Young Adults with PWS (12 20.99 Y ears ) Teenagers and adults with PWS (12 20.99y) had significantly higher ghrelin levels than normal lean controls ( p=0.011 ) and EMO subjects ( p=0.0085 ) of the same age [Figure 3 4] They also had significantly elevated leptin levels ( p=0.0094 ) and BMI Z scores relative to normal lean controls [Table 3 2 ] EMO teenagers and young adults had ghrelin levels similar to normal lean controls ( p=0.49 ) but significantly elevated leptin leve ls relative to b oth PWS and normal lean control subjects ( p=0.0060** & p<0.001* respectively). H yperghrelinemia Precedes Obesity and H yperphagia in PWS The nutritional phases in PWS were more predictive of ghrelin levels in PWS individuals than was age or any other factor. I ndividuals in nutr itional phase 1a, which is characterized by poor appetite and failure to thrive, had the highest ghrelin levels relative to all the other nutritional phases combined [Figure 3 5]. Nutritional phase 3, which is charac terized by hyperphagia and obesity, had some of the lowest observed ghrelin levels. Correlation of Ghrelin with Leptin, BMI z Score, DEXA, and Growth H o rmone T herapy in PWS PWS individuals on growth hormone therapy had serum ghrelin levels about 1202 534 pg/ml less than individuals not on growth hormone therapy [Figure 3 6]. This difference was significant after adjusting for age and sex ( p=0.043 ). Ghrelin correlated significantly with leptin in normal control infants and young children 0 1.99 y ears old ( p=0.0063** ) but not in PWS children ( p=0.98 ) of the same age. However, leptin correlated significantly with weight for length in PWS infants and

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32 young children 0 1.99 years old ( p=0.0025* ) but not in normal control infants and young children ( p=0.90 ) o f the same age [Table 3 3 ]. Above the age of 2 years, g hrelin did not significantly correlate with leptin, BMI z score or DEXA in PWS ( p=0.74 ), normal lean controls ( p=0.089 ), and EMO subjects ( p=0.71 ) [Table 3 4 ]. However leptin correlated significantly with DEXA and BMI z score in PWS ( p<0.001** & p=0.0013** respectively ) and norma l lean control subjects ( p<0.001 & p=0.0013** respectively). DEXA correlated with BMI z score in all three groups (PWS, p<0.001** ; Sib.C, p<0.001** ; EMO, p=0.0082** ) [Table 3 4 ] There was no significant difference in ghrelin levels of PWS individuals with Type 1 or Type 2 deletions ( p=0.67 ) or between UPD and deletion PWS subjects ( p=0.46 ). The significance of this work in relationship to the current state of the fi eld is discussed in chapter 6. The ghrelin assay used in this study had intra assay variability less than 4% and an inter assay variability less than 15%.

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33 Table 3 1 List of subjects, observations and characteristics PWS Sib.C EMO Age group 1 0 1.99 years 0 1.99 years 0 1.99 years Subjects 18 (9M, 9F) 12 (7M, 5F) N/A Observations 25 (15M, 10F) 14 (8M, 6F) N/A Average age (years) 1.1 0.5 0.91 0.5 N/A Mol. Class (Del/UPD/ID) 14/9/2 N/A N/A GH treatment (Yes/No) 14/11 N/A N/A Age group 2 2 4.99 years 2 4.99 years 2 4.99 years Subjects 41 (22M, 19F) 26 (11M, 15F) 9 (6M, 3F) Observations 53 (27M, 26F) 28 (11M, 17F) 9 (6M, 3F) Average age (years) 3.7 0.7 3.7 0.9 4.1 0.9 Mol. Class (Del/UPD/ID) 33/18/2 N/A N/A GH treatment (Yes/No) 49/4 N/A N/A Age group 3 5 11.99 years 5 11.99 years 5 11.99 years Subjects 29 (12M, 17F) 54 (25M, 29F) 20 (10M, 10F) Observations 41 (17M, 24F) 74 (31M, 43F) 28 (16M, 12F) Average age (years) 7.5 1.8 8.0 1.7 8.2 1.8 Mol. Class (Del/UPD/ID) 26/13/2 N/A N/A GH treatment (Yes/No) 36/5 N/A N/A Age group 4 12 20.99 years 12 20.99 years 12 20.99 years Subjects 12 (7M, 5F) 23 (14M, 9F) 12 (5M, 7F) Observations 17 (9M, 8F) 31 (18M, 13F) 15 (7M, 8F) Average age (years) 16.2 2.8 15.5 2.0 15.7 2.7 Mol. Class (Del/UPD/ID) 15/2/0 N/A N/A GH treatment (Yes/No) 14/3 N/A N/A Age is expressed as Mean SD; N/A = Not Applicable ; M = Male; F = Female Sib.C = Normal weight Sibling C ontrols PWS = Prader Willi Syndrome EMO = Early onset Morbid O besity G H treatment = Growth H ormone treatment Molecular class and GH treatment are descriptive characteristics for observations

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34 Table 3 2 Results of biological assays and measurements PWS Sib.C EMO P1 P2 P3 Age group (years) 0 1.99 0 1.99 0 1.99 Ghrelin (pg/ml) 5521 3696 2883 1172 N/A 0.016* N/A N/A Leptin (pg/ml) 272 231 216 145 N/A 0.48 N/A N/A Weight for length 25.07 28.17 57.41 37.50 N/A 0.025* N/A N/A DEXA 21.36 +/ 7.89 19.64 6.33 N/A 0.57 N/A N/A Age group (years) 2 4.99 2 4.99 2 4.99 Ghrelin 3113 1898 2556 927 3430 2320 0.12 0.71 0.30 Leptin 1389 1785 150 99 2248 1107 <0.001** 0.098 <0.001** BMI z score 0.93 1.55 0.32 1.19 4.29 0.79 0.074 <0.001** <0.001** DEXA 24.98 10.47 18.61 6.39 44.04 5.78 0.005** <0.001** <0.001** Age group (years) 5 11.99 5 11.99 5 11.99 Ghrelin (pg/ml) 2476 1332 2111 1013 1645 983 0.21 0.021* 0.10 Leptin (pg/ml) 2107 1572 397 720 2408 1569 <0.001** 0.56 <0.001** BMI z score 1.62 1.17 0.35 0.92 2.72 0.22 <0.001** <0.001** <0.001** DEXA 35.08 12.75 20.39 8.05 45.83 4.97 <0.001** <0.001** <0.001** Age group (years) 12 20.99 12 20.99 12 20.99 Ghrelin (pg/ml) 2086 885 1233 509 1053 847 0.011* 0.0085 ** 0.49 Leptin (pg/ml) 2837 1839 1138 1485 5459 2289 0.0094** 0.0060** <0.001** BMI z score 2.10 0.66 0.50 1.08 2.73 0.34 <0.001** 0.0051** <0.001** DEXA 47.95 8.72 26.00 10.79 54.28 6.09 <0.001** 0.049* <0.001** All data expressed as Mean SD ; = p <0.05, ** = p <0.01 P1 = p value for comparison of PWS vs Sib.C P2 = p value for comparison of PWS vs EMO P3 = p value for comparison of Sib.C vs EMO Sib.C = Normal weight Sibling Controls PWS = Prader Willi Syndrome EMO = Early onset Morbid Obesity

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35 Table 3 3 Mean change in Dependent V ariable (DV) per Unit Change in Independent Variable (IV): Age 0 1.99: Slope (SE)[N]{P value} DV IV PWS Sib. C Ghrelin Leptin 0.09(4.7)[14]{0.98} 7.2(1.6)[7]{0.0063} Ghrelin Weight for Length 17.6(34.5)[16]{0.62} 6.6(12.9)[9]{0.62} Ghrelin Dexa 110.5(138.4)[15]{0.44} 66.6(59.2)[6]{0.32} Leptin Dexa 13.4(7.21)[14]{0.088} 5.39(8.28)[7]{0.54} Leptin Weight for Length 4.55(1.80)[15]{0.025} 0.19(1.37)[9]{0.90} Dexa Weight for Length 0.17(0.058)[16]{0.0096} 0.086(0.053)[8]{0.16}

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36 Table 3 4 Mean ch ange in Dependent V ariable (DV) per Unit Change in Independent Variable (IV): Age 2+: Slope (SE)[N] {P value} DV IV PWS Sib.C EMO Ghrelin Leptin 0.032(0.094)[83]{0.74} 0.23(0.13)[87]{0.089} 0.034(0.087)[40]{0.71} Ghrelin BMI Z 142.1(123.8)[118]{0.26} 36.9(92.4)[135]{0.69} 227.1(251.7)[52]{0.38} Ghrelin DEXA 13.2(15.4)[106]{0.40} 9.2(11.6)[122]{0.42} 51.5(30.2)[44]{0.12} Leptin DEXA 74.2(16.0)[86]{<0.001} 61.0(8.8)[85]{<0.001} 263(38.1)[39]{<0.001} Leptin BMI Z 664(123)[100]{<0.001} 352(85.8)[94]{0.0013} 692(418)[46]{0.13} DEXA BMI Z 5.69(0.54)[120]{<0.001} 5.17(0.61)[127]{<0.001} 3.70(1.17)[50]{0.0082}

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37 Figure 3 1 Box plot of ghrelin levels in infants and young PWS and normal control (Sib.C) children 0 1.99 years old. The tails of the box plot represent the 10 th and 90 th pe rcentiles; the bars repres ent the 25 th 50 th and 75 th percentiles. The dots represent outlie rs.

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38 Figure 3 2 Box plot of ghrelin levels in PWS Sib.C and EMO children 2 4.99 years old. The tails of the box plot represent the 10 th and 90 th percentiles; th e bars repres ent the 25 th 50 th and 75 th percentiles. The dots represent outliers.

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39 Figure 3 3 Box plot of ghrelin levels in PWS, Sib.C and EMO children 5 11.99 years old. The tails of the box plot represent the 10 th and 90 th percentiles; the horizontal bars are the 25 th 50 th and 75 th percentiles. The dots represent outliers.

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40 Figure 3 4. Ghrelin is elevated in teenagers and adults with PWS. Box plot of ghrelin levels in PWS, Sib.C and EMO teenagers and young adults 12 20 .99 years old. The tails of the box plot represent the 10 th and 90 th percentiles; the bar s are the 25 th 5 0 th and 75 th percentiles. The dots represent outliers.

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41 Figure 3 5. Bar chart of average ghrelin levels in PWS nutritional phases Nutritional phase is more prognostic of ghrelin levels in PWS.

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42 Figure 3 6. Growth hormone therapy decreases ghrelin l evels in PWS Bar chart of mean ghrelin levels of PWS individuals not on growth hormone therapy (PWS No GH) versus PWS individuals on growth hormone therapy (PWS GH)

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43 A B Figure 3 7. PWS children have low weight for length and normal body fat ; A) Box plot of weight for length percentile in PWS children relative to normal control children (0 1.99 y ) ; B) Box plot of percent body fat in PWS children relative to normal controls (0 1.99y) The tails of the box plots represent the 10 th and 90 th perce ntiles; the bars are the 25 th 50 th and 75 th percentiles. The dots represent outliers.

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44 CHAPTER 4 ANALYSIS OF LEPTIN SIGNALING IN INDIVIDUALS WITH PRADER WILLI SYNDROME THROUGH ANA LYSIS OF CIRCULATING ALPHA MELANOCYTE STIMULATI NG HORMONE LEVE LS Hy pothalamic Leptin Signaling I s U nknown in PWS Th e hypothalamic leptin melanocortin signaling pathway consists of leptin and the melanocortin peptides POMC, alpha MSH and MC4R Leptin signals through its receptor in the brain to up regulate POMC mRNA expression and translation in the hypothalamus. T he POMC peptide is processed to release a lpha MSH, which activates the melanocortin 4 receptor (MC4R) leading to decreased appetite and increased energy expenditure. Little is known about leptin levels in the hypothalamus of individuals with PWS Obese adults with PWS have elevated peripheral leptin levels that are appropriate for their degree of obesity and fall well within the range of the normal obese population (Butler et al., 1998) However, while d irect delivery of leptin into the brain has been shown to reduce feeding, peri p heral injection of leptin fails to reduce feeding and does not significantly alter leptin levels in the cerebrospinal fluid (Schw artz et al., 1996; Van Heek et al., 1997; Ramsey et al., 1998) Thus it remains possible that PWS subjects may suffer from inadequate hypothalamic leptin signaling despite high peripheral leptin levels. The aim of this study was to analyze hypothalamic leptin signaling in individuals with Prader Willi syndrome by measuring circulating alpha MSH levels Because the brain is the major source of circulating alpha MSH in the body, w e reasoned that periph eral changes in alpha MSH level would reflect changes in anorexigenic leptin signaling in the brain. We hypothesize d that if hypothal amic leptin signaling was

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45 appropriate in PWS, then ob ese and hyperphagic children with PWS will have circulating alpha MSH levels significantly higher than normal lean control children To test this hypothesis we measured plasma leptin levels and serum alpha MSH levels in normal lean control children (Sib.C ) in non PWS children with e arl y onset morbid obesity (EMO), in PWS children before the onset o f obesity and hyperphagia ( PWS BOH ) and in PWS children after the onset o f obesi ty and hyperphagia ( PWS AOH) [Table 4 1] Multiple s erum and plasma samples were obtai ned more than once from subjects wi th repeated visits to our clinic thus accounting for the discrepancy between subjects and samples (observations) in Table 4 1. L eptin I s A ppropriately Elevated in O be se Hyperphagic PWS C hildren We used the LUMINEX assay system (Millipore Inc., CA, USA) to measure plasma leptin levels in 21 non obese children with PWS (PWS BOH) 17 obese PWS children (PWS AOH), 17 normal lean control children (Sib.C) and 11 children with early onset morbid (EMO) [Table 4 1] Peripheral leptin levels in non obese non hyperphagic PWS children (PWS BOH) did not differ significantly from normal lean con trol children ( 194.9 194.9 pg/ml vs 222.5 163.0 pg/ml; p= 0.64 ) but was significantly lower relative to leptin levels in obese hyperphagic PWS children ( 194.9 194.9 pg/ml vs 2464 1475 pg/ml; p<0. 001 ** ) and EMO children ( 194.9 194.9 pg/ml vs 2817 1737 pg/ml; p<0.0 01 ** ) [Figure 4 1] O bese hyperphagic PWS children had significantly elevated plasma leptin relative to normal lean control children ( 2464 1475 pg/ml vs 194.9 194.9 pg/ml; p<0.0 01 ** ) however their leptin did not differ signif icantly from leptin levels in EMO children ( 2464 1475 pg/ml vs 2817 1737 pg/ml; p=0.55 ).

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46 Leptin levels were significantly elevated in EMO children relative to normal lean control children ( 2817 1737 pg/ml vs 194.9 194.9 pg/ml; p<0.0 01 ** ). A lpha MSH Is Not E levated in O bese H yperphagic PWS C hildren I us ed a competitive ELISA assay (Phoenix Pharmaceuticals Inc., USA) to measure serum alpha MSH levels in the same group of 21 non obese PWS children, 17 obese PWS children, 17 normal weight control childr en, and 11 children with early onset morbid described above. Obese PWS children had peripheral a lpha MSH levels similar to normal lean control children ( 209.6 85.09 pg/ml vs 216 .1 75.34 pg/ml; p=0.82 ) but significantly lower than non obese PWS children ( 209.6 85.09 pg/ml vs 282.7 111.9 pg/ml; p=0.033 ) and EMO children ( 209.6 85.09 pg/ml vs 5883 1137 pg/ml; p=0.048 ) [Figure 4 2] Non obese non hyperphagic PWS children had significantly higher peripheral alpha MSH levels than normal lean control children ( 282.7 111.9 pg/ml vs 216.1 75.34 pg/ml; p=0.043 ). However their serum alpha MSH was significantly lower relative to EMO subjects ( 282.7 111.9 pg/ml vs 5883 1137 pg/ml; p=0.029 ) [Figure 4 2] EMO children had significantly elevated alpha MSH levels than normal lean control children ( 5883 1137 pg/ml vs 216.1 75.34 pg/ml; p=0.048 ) [Figure4 2].

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47 Table 4 1. Leptin and alpha MSH levels in PWS, Sib.C & EMO subjects Group Subjects Observations Age (years) BMI Z Leptin (pg/ml) MSH (pg/ml) PWS BOH 5M, 4F 13M, 8F 2.0 1.1 0.58 0.99 194.9 194.9 282.7 111.9 PWS AOH 5M, 5F 8M, 9F 7.1 2.9 2.4 0.47 2464 1475 209.6 85.09 Sib.C 3M, 4F 6M, 11F 5.5 3.9 0.44 0.57 222.5 163.0 216.1 75.34 EMO 8M, 3F 10M, 4F 6.6 3.0 3.4 0.99 2817 1737 5883 1137 Data are expressed as Mean SD

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48 Figure 4 1 Box plot of peripheral leptin levels in obese hyperphagic PWS children (PWS AOH), non obese non hyperphagic PWS children (PWS BOH) normal control children (Sib.C) and children with early onset morbid obesity (EMO ) The tails of the box plot represent the 10 th and 90 th percentiles; the bars are the 25 th 50 th and 75 th percentiles. The dots represent outliers.

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49 Figure 4 2. Box plo t of peripheral alpha MSH levels in obese PWS (PWS AOH) and non obese PWS (PWS BOH) children, normal weight control children (Sib.C) and children with ea rly onset morbid obesity (EMO) The tails of the box plot represent the 10 th and 90 th percentiles; the bars are the 25 th 50 th and 75 th percentiles.

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50 CHAPTER 5 ANALYSIS OF CIRCULAT ING BRAIN DERIVED NEUROTROPHIC FACTOR LEVELS IN INDIVIDUALS WITH PRADER WILLI SYNDROME Brain D erived Neurotrophic Factor and Prader Willi Syndrome Brain derived neurotrophic factor (BDNF) is a neuropeptide that is highly expressed in the brain and plays an important role in energy homeostasis. BDNF functions downstream of the hypothalamic leptin melanocortin signaling pathway and modulates food intake and energy expenditure. BDNF haploinsufficiency is associated with low peripheral BNDF levels severe hyperphagia, obesity, and developmental delay (Han JC et al 2008; Gray J et al 2006) Mutations in the BDNF receptor TrkB result in morbid obesity and hyperpha gia. Since BDNF is produced primarily in the brain, p eripheral BDNF concentrations are thought to reflect cerebral BDNF output A recent pilot study reported low peripheral BDNF levels in 13 obese PWS patients relative to obese and lean control subjects suggesting insufficient centra l BDNF production in individuals with PWS (Han et al., 2010). In this study, we investigate serum BDNF levels in a larger cohort of obese PWS subjects { 28 subjects 47 observations ; age 10.5 7 .5yr; BMI Z, 2.6 0.5 } in lean control subjects (Sib.C) { 66 subjects 81 obeservations ; age, 9.1 6.1yr; BMI Z, 0.06 0.7 } and in non PWS individuals with early onset morbid obesity (EMO) { 36 subjects 48 observations ; age, 10.8 5.8yr; BMI Z, 3.0 0.7} [ Table 5 1; Figure 5 1] Serum BDNF Is E levated in PWS S ubjects PWS subjects as a group had significantly higher m ean serum BDNF levels relative to lean control subjects ( 9846 7894 pg/ml vs 5623 7808 pg/ml; p=0.001 1 ** ) However, their serum BDNF concentration was not s ignificantly different from levels in non PWS individuals with early onset morbid obesity ( 9846 7894 pg/ml vs 7808

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51 8135 pg/ml; p=0.22 ). EMO subjects did not have significantly higher serum BDNF levels relative to lean controls ( 7808 8135 pg/ml vs 56 23 7808 pg/ml, p=0.089 ) [Figure 5 2]. There was no significant correlation between BDNF and age in PWS s ubjects ( r = 0.05248; p=0.7261 ), in lean controls ( r = 0.1842; p=0.082 ) or in EMO subjects ( r = 0.04125; p=0.78 ). There was also no significant correlation with BMI z scores in PWS, Sib.C or EMO subjects ( p=0.75, 0.19 & 0.68 respectively ). However, serum BDNF levels correlated signi ficantly with nutritional phase in obese PWS subjects ( r = 0.3243; p=0.026 ). B DNF L evels D ecrease with Onset of H yperphagic N utritional P hases in PWS To assess the effect of PWS nutritional phases on circulating BDNF levels in PWS subjects I analyzed serum BDNF level s of obese PWS subjects in nutritional phase 2a, 2b and 3. Serum BDNF level s decreased from 12643 3280 pg/ml in nutri tional phase 2a, to 11018 2012 pg/ml in nutritional phase 2b and then to 7823 1550 pg/ml in nutritional phase 3. Serum BDNF of PWS indi viduals in nutritional phase 2a was significantly elevated relativ e to lean controls (12643 3280 pg/ml vs 5623 691 pg/ml; p=0.0091** ). PWS individuals in nutritional phase 2b also had significantly elevated serum BDNF levels relative to lean controls (1108 5012 pg/ml vs 5623 691 pg/ml; p=0.0050** ). However serum BDNF level of PWS subjects in nutritional phase 3 was not significantly higher than BDNF levels in lean controls ( 7823 1550 pg/ml vs 5623 691 pg/ml; p=0.17 ) [Figure 5 3]

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52 Table 5 1. S ubjects, observations and biological v alues for BDNF analysis Group Subjects Observations Age (yr) BMI Z BDNF P1 P2 P3 PWS 16M, 12F 25M, 22F 10.5 7.5 2.6 0.5 9846 7894 0.0011** 0.22 0.089 Sib.C 30M, 36F 40M, 36F 9.1 6.1 0.06 0.7 5623 6561 EMO 17M, 19F 25M, 23F 10.8 5.8 3.0 0.7 7808 8135 Data are expressed as Mean test. ( P1= PWS vs Sib.C; P2 = PWS vs EMO; P3 = Sib.C vs EMO )

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53 A B Figure 5 1. G raphical representation of subject ages and BMI z scores; A) Scatter plot of ages with Mean ; B) Scatter plot of BMI z s cores with Mean

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54 Figure 5 2. Box plot of seru m BDNF levels in PWS, Sib.C and EMO subjects. The tails of the box plot represent the 10 th and 90 th per centiles; the bars are the 25 th 50 th and 75 th percentiles. The dots represent outliers.

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55 Figure 5 3. BDNF levels decrease with onset of hyperphagia in PWS. Box plot of serum BDNF lev els in PWS nutritional phases 2a, 2b, and 3 and also in normal sibling control subjects of similar ages The tails of the box plot represent the 10 th and 90 th percentiles; the bars are the 25 th 50 th and 75 th percentiles. The dots represent outliers.

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56 CHAPTER 6 DISCUSION Hype rghrelinemia Begins Early i n Prader Willi Syndrome I measured serum ghrelin levels in a young er and larger group of PWS subjects and normal control subjects (2 months to 20.99 years) than has been previously analyzed by others [Table 3 1] PWS infants and young children between 2 months 2 years had significantly elevated ghrelin levels than their normal counter part s of similar age while the ghrelin of PWS children between 2 12 years was not significantly elevated than their normal counterparts within the same age group However, w e found that PWS nutritional phase was more prognostic of ghrelin levels in PWS subjects than age or BMI Z. The onset of the hyperphagic nutritional phases in PWS patients correlated significantly with lower ghrelin levels: PWS children in nutritional phase 1a and 1b had significantly elevated ghrelin le vels relative to normal control children of same age while PWS individuals in nutritional phase 3 did not have significantly elevated ghrelin levels relative to normal childre n of same age Given that the age of onset of the nutritional phases varies among individuals with PWS, analysis o f ghrelin levels in PWS by age alone may be misleading. Thus the inconsistencies in the literature on ghrelin levels in young PWS children m ay be due to a greater number of PWS subjects in the more advanced nutritional phases in the study group Given that ghrelin l evels were the highest in PWS children with poor appetite (Phase 1a) it seems unlikely that elevated ghrelin levels are responsib le for the switch to the hyperphagic phases of PWS. However, i t has been demonstrated in mice that ghrelin can act to increase fat mass independent of its effect on appetite (Perez Tilve et al., 2011) It is therefore likely that the elevated ghrel in levels are causing the increased

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57 fat mass seen in infants with PWS compared to normal infants with similar body mass indices (BMI). This would explain why PWS infants have normal body fat even though they surfer from failure to thrive and have signific antly lower weight for length percentile relative to normal infants. We also observed that PWS individuals on growth hormone therapy had significantly lower ghrelin levels than PWS individuals not on growth hormone therapy Thus part of the effect of gr ow th hormone in promoting lean body mass in young PWS subjects may result from its ability to significantly decrease ghrelin levels early on PWS Individuals May Suffer F rom Leptin Resistance Hypothalamic leptin signals through its receptor to induce POMC mRNA expression in the brain consequently leading to increased alpha MSH levels both centrally and peripherally My data demonstrates that non obese PWS subjects have significantly elevated serum alpha MSH levels relative to normal lean controls even th ough their plasma leptin levels are not elevated. However, obese PWS subjects have significantly lower alpha MSH levels than non obese PWS subje cts even though their plasma lepin is significantly elevated Thus it appears serum alpha MSH levels decrease with onset of obesity and hyperphagia in PWS. Leptin levels in the cerebro spinal fluid (CSF) correlate strongly with peripheral leptin levels in a nonlinear manner and also with body mass index (Schwartz et al., 1996) It is thought that plasma leptin enters human CSF in proportion to body adiposity, however the efficiency of this process is significantly lower in obese individuals with high peripheral leptin levels. Schwartz et al hypothesized that a saturable mechanism mediates CSF leptin transport and that leptin resistance may

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58 occur in ob ese individuals with high peripheral leptin le vels due to reduced efficiency of brain leptin delivery I t is possible that the high peripheral leptin levels of PWS subjects have triggered a saturable mechanism, resulting in leptin resistance through inefficient leptin transport into the brain Decreased BDNF levels in obese PWS subjects appear to support this hypothesis. BDNF function s dow nstream of the leptin melanocortin signaling pathway (Xu et al., 2003) ; rough alpha MSH induce s BDNF expression through the cyclic AMP protein kinase A pathway (Caruso et al., 2012) J ust like with al pha MSH, peripheral BDNF levels decrease with onset of obesity and hyperphagia in PWS. W e hyp othesize that elevated ghrelin levels early on in PWS infants leads to increased adiposity, which leads to elevated leptin levels in older children with PWS that trigger lept in resistance, leading to inadequate hypothalamic leptin melanocortin signaling an d low BDNF levels, resulting in development of obesity and hyperphagia in PWS children and adults [Figure 6 1]

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59 Figure 6 1. Working model for development of obesity and hyperphagia in PWS. Elevated ghrelin levels in PWS infants activate lipogenic genes early on, leading to increased adiposity and early onset obesity. Consequently, peripheral leptin levels are elevated significantly, triggering a saturable mechanism that decreases the efficiency of leptin uptake by the brain (leptin resistance). Low brain leptin levels results in decreasd alpha MSH and BDNF levels, which further promote obesity and trigger hyperphagia in PWS subjects.

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60 CHAPTER 7 FUTURE DIRECTIONS Analysis of POMC and PRCP E xpression in PWS We will further analyze alpha MSH levels in the different nutritional phases in Prader Willi syndrom e with larger sample sizes. To determine whether changes in alpha MSH levels in PWS stem from transcription al regulation or regulation of the mature protein, we will evaluate POM C mRNA expression in each nutritional phase. The enzyme prolylcarboxypeptidase ( PRCP ) degrades and regulates alpha MSH protein levels and f unction and has been recently implicated in obesity. We will analyze expression of PRCP mRNA and protein levels in PWS individuals at different nutritional phases Analysis of Ghrelin Responsive Pathways in Young Children with PWS The inability of infants and young chi ldren with PWS to respond to high ghrelin levels may be the result of poorly developed ghrelin respon sive pathways early on. To this end, we intend to measure ghrelin responsive proteins such as oxytocin, neuropeptide y and agouti related protein in infants and young children with PWS. We will also analyze frozen adult PWS brain samples for expression of ghrelin responsive neurons in the hypothalamus by immunohistochemistry.

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64 MacQueen, G.M., Ramakrishman, K., Croll, S.D., Siuciak, J.A., Yu, G., and Fahnestock, M. (2001). Performance of heterozygous brain derived neurotrophic factor knockout mice on behavioral analogues of anxiety, nociception, and depression. Behavioral Neuroscience 115 1145. Miller, J.L., Lynn, C.H., Driscoll, D.C., Goldsto ne, A.P., Gold, J. A., Kimonis, V., Dykens, E., Butler, M.G., Shuster, J.J., and Driscoll, D.J. (2011). Nutritional phases in Prader Willi syndrome. Am J Med Genet 155 1040 1049. Mohammad, S., Baldini, G., Granell, S., Narducci, P., Martelli, A.M., and Ba ldini, G. (2007). Constitutive traffic of melanocortin 4 receptor in Neuro2A cells and immortalized hypothalamic neurons. J Biol Chem 282 4963 4974. Nakagawa, T., Tsuchida, A., Itakura, Y., Nonomura, T., Ono, M., Hirota, F., Inoue, T., Nakayama, C., Taiji M., and Noguchi, H. (2000). Brain derived neurotrophic factor regulates glucose metabolism by modulating energy balance in diabetic mice. Diabetes 49 436 444. Nicholson, J., Peter, J.C., Lecourt, A.C., Barde, Y.A., and Hofbauer, K. (2007). Melanocortin 4 Receptor Activation Stimulates Hypothalamic Brain Derived Neurotrophic Factor Release to Regulate Food Intake, Body Temperature and Cardiovascular Function. Journal of Neuroendocrinology 19 974 982. Nijenhuis, W.A.J., Oosterom, J., and Adan, R.A.H. (200 1). AgRP (83 132) acts as an inverse agonist on the human melanocortin 4 receptor. Mol Endocrinol 15 164 171. Ollmann, M.M., Wilson, B.D., Yang, Y.K., Kerns, J.A., Chen, Y., Gantz, I., and Barsh, G.S. (1997). Antagonism of central melanocortin receptors i n vitro and in vivo by agouti related protein. Science 278 135 138. Oswal, A., and Yeo, G.S.H. (2007). The leptin melanocortin pathway and the control of body weight: lessons from human and murine genetics. Obesity Reviews : an Official Journal of the Int ernational Association for the Study of Obesity 8 293 306. Perez Tilve, D., Heppner, K., Kirchner, H., Lockie, S.H., Woods, S.C., Smiley, D.L., Tschop, M., and Pfluger, P. (2011). Ghrelin induced adiposity is independent of orexigenic effects. Faseb J 25 2814 2822. Proto, C., Romualdi, D., Cento, R.M., Romano, C., Campagna, G., and Lanzone, A. (2007). Free and total leptin serum levels and soluble leptin receptors levels in two models of genetic obesity: the Prader Willi and the Down syndromes. Metab Clin Exp 56 1076 1080. Pruunsild, P., Kazantseva, A., Aid, T., Palm, K., and Timmusk, T. (2007). Dissecting the human BDNF locus: bidirectional transcription, complex splicing, and multiple promoters. Genomics 90 397 406.

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65 Ramsey, J.J., Kemnitz, J.W., Colman R.J., Cunningham, D., and Swick, A.G. (1998). Different Central and Peripheral Responses to Leptin in Rhesus Monkeys: Brain Transport May Be Limited. J Clin Endocrinol Metab 83 3230 3235. Rios, M., Fan, G., Fekete, C., Kelly, J., Bates, B., Kuehn, R., Lechan, R.M., and Jaenisch, R. (2001). Conditional deletion of brain derived neurotrophic factor in the postnatal brain leads to obesity and hyperactivity. Mol Endocrinol 15 1748 1757. Roth, C.L., Ludwig, M., Woelfle, J., Fan, Z. C., Brumm, H., Biebermann, H., and Tao, Y. X. (2009). A novel melanocortin 4 receptor gene mutation in a female patient with severe childhood obesity. Endocr 36 52 59. Schwartz, M.W., Peskind, E., Raskind, M., Boyko, E.J., and Porte, D. (1996). Cerebrospinal fluid leptin levels: relationship to plasma levels and to adiposity in humans. Nature Medicine 2 589 593. Shimizu, H., Inoue, K., and Mori, M. (2007). The leptin dependent and independent melanocortin signaling system: regulation of feeding and energy expenditure. J Endocrinol 193 1 9. Shinyama, H., Masuzaki, H., Fang, H., and Flier, J.S. (2003). Regulation of melanocortin 4 receptor signaling: agonist mediated desensitization and internalization. Endocrinology 144 1301 1314. Steculorum, S.M., and Bouret, S.G. (2011). Developmental effects of ghrelin. Peptides 32 2362 2366. Tan, T.M. M., Vanderpump, M., Khoo, B., Patterson, M., Ghatei, M.A., and Goldstone, A.P. (2004). Somatostatin infusion lowers plasma ghreli n without reducing appetite in adults with Prader Willi syndrome. J Clin Endocrinol Metab 89 4162 4165. Unger, T.J., Calderon, G.A., Bradley, L.C., Sena Esteves, M., and Rios, M. (2007). Selective deletion of Bdnf in the ventromedial and dorsomedial hypot halamus of adult mice results in hyperphagic behavior and obesity. J Neurosci 27 14265 14274. Van Heek, M., Compton, D.S., France, C.F., Tedesco, R.P., Fawzi, A.B., Graziano, M.P., Sybertz, E.J., Strader, C.D., and Davis, H.R. (1997). Diet induced obese m ice develop peripheral, but not central, resistance to leptin. J Clin Invest 99 385 390. Xu, B., Goulding, E.H., Zang, K., Cepoi, D., Cone, R.D., Jones, K.R., Tecott, L.H., and Reichardt, L.F. (2003). Brain derived neurotrophic factor regulates energy bal ance downstream of melanocortin 4 receptor. Nat Neurosci 6 736 742. Yang, Y., Thompson, D.A., Dickinson, C.J., Wilken, J., Barsh, G.S., Kent, S.B.H., and Gantz, I. (1999). Characterization of Agouti related protein binding to melanocortin receptors. Mol E ndocrinol 13 148 155.

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66 Yeo, G.S.H., Connie Hung, C. C., Rochford, J., Keogh, J., Gray, J., Sivaramakrishnan, S., O'rahilly, S., and Farooqi, I.S. (2004). A de novo mutation affecting human TrkB associated with severe obesity and developmental delay. Nat N eurosci 7 1187 1189.

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67 BIOGRAPHICAL SKETCH Frederick A. Kweh was born in Cameroon, West Africa. He moved to the United States of America in the fall of 1999 to live with his uncle Andrew Kweh in Florida. From the fall of 2000 to the spring of 2002 he attended Santa Fe Community college in Gainesville, during which time he was a student ambassador for the college. He graduated with an Associate of Arts degr ee in the spring of 2002 and was adm itted at the University of Florida as a junior for the 2002 fall semester. He graduated with a Bachelor of Science degree in m icrobiology in May 2004 after which he worked as a biological technician in the laboratory of Dr. Margaret Wallace. In the fall o f 200 6 he began his PhD work in the I nterdiscipl inary Program in Biomedical Science at the University Of Florida College Of Medicine His graduate research in the Medical Scienc es was done under the m entorship of Dr. Daniel J. Driscoll.