Ronald M. Lechan, M.D, Ph.D - US grants

The main objectives of this study are to identify the hormones, peptides and neurotransmitters that regulate biosynthesis of TRH in the paraventricular nucleus (PVN) of the hypothalamus, the principal source of neurons that regulate anterior pituitary TSH secretion, and to elucidate their mechanisms of action. The factors responsible for the cell-specific feedback effects of thyroid hormone (TH) on proTRH mRNA in the PVN will be investigated by nuclear run-off assays to determine whether TH regulates the rate of transcription and/or stability of proTRH mRNA; quantitating the amount of (125I)T3 produced from (125I)T4 in the PVN to determine whether TH effects are dependent upon local 5'- monodeiodination of T4 or exerted directly by circulating levels of T3; and in situ hybridization and immunocytochemistry using c- erbA probes as markers of the TH receptor to determine if TH has direct or indirect effects on TRH-synthesizing neurons. The origin and identification of peptides and neurotransmitter substances which make synaptic contacts on TH-responsive PVN neurons will be determined by double immunolabeling techniques including anterograde tracing with PHA-L and immunocytochemistry at light and ultrastructural levels. The role of each of these factors (agonists and/or antagonists) in regulating TRH gene expression will be demonstrated in vivo after stereotaxic implantation unilaterally into the hypothalamus using semiquantitative in situ hybridization, immunocytochemistry and computer image analysis. Similar techniques will be used to determine whether the nyctohemeral variation of TSH secretion and changes in TH levels associated with glucocorticoids, cold exposure and caloric deprivation are mediated through effects on proTRH mRNA and whether TH establishes the permanent set point for TRH secretion at a critical time during development. The significance and functional regulation of another group of TRH-synthesizing. TH-unresponsive PVN neurons will also be determined by retrograde and anterograde tracing studies and immunocytochemistry to demonstrate the topography of innervation of the median eminence, its afferent input and neurotransmitter/neuropeptide mediators.

[unreadable] DESCRIPTION (provided by applicant): Thyrotropin-releasing hormone (TRH) produced by neurons in the hypothalamic paraventricular nucleus (PVN) has an important role in the regulation of energy homeostasis by establishing circulating levels of thyroid hormone under normal conditions and during special circumstances when metabolic adaptation is necessary such as fasting, infection and chronic illnesses. Mechanisms whereby metabolic signals regulate TRH neurons in the PVN will be explored, facilitated by the use of the Cre/loxP recombinase system in transgenic mice in which Cre-recombinase is expressed selectively in TRH cells. Attention will be given to the importance of direct vs indirect leptin signaling during fasting and in association with diet-induced obesity, the role of AGRP as an inverse agonist at MC-4 receptors, participation of CREB, ERK and AMPK signaling pathways as mediators of leptin's diverse actions on hypophysiotropic TRH neurons, and cannabinoid/glutamate interactions as a novel mechanism involved in fasting-induced suppression the HPT axis. The importance of the hypothalamic dorsomedial nucleus (DMN) as a relay station between the arcuate nucleus and TRH neurons in the PVN will be explored and the hypothesis tested that the DMN integrates signals from leptin- responsive arcuate nucleus neurons and the subparaventricular zone to give rise to the circadian periodicity of the HPT axis. Mechanisms by which endotoxin suppress hypophysiotropic TRH neurons will also be studied, focusing on effects mediated by cAMP-response element modulator (CREM), inducible cAMP early repressor (ICER), corticotropin-releasing hormone (CRH) and the role of increased T3 levels in the mediobasal hypothalamus as a result of endotoxin-induced increased type 2 iodothyronine deiodinase levels in tanycytes. Thyrotropin-releasing hormone (TRH) plays a major role in energy homeostasis by establishing circulating levels of thyroid hormone under normal conditions and during special circumstances such as fasting and critical illness when changes in thyroid status are required for adaptation. The proposed studies will elucidate how this specialized group of neurons in the hypothalamic paraventricular nucleus is regulated by metabolic signals and the neuroanatomical pathways and modulators involved, providing insight into disorders commonly referred to as the nonthyroidal illness syndrome. [unreadable] [unreadable] [unreadable]

The long term goal of this study is to better understand the physiologic mechanisms and chemical mediators responsible for opiate withdrawal. We have observed that a unique population of neurons located in the ventrolateral region of the midbrain periaqueductal gray (PAG) show a marked increase (approximately 500%) in pro-thyrotropin-releasing hormone (proTRH) gene expression during opiate withdrawal. Since excitation of this region evokes a quiescent and hyporeactive pattern of behavior, it is hypothesized that proTRH neurons in the ventrolateral PAG are activated as a compensatory response to the hyperactive state of opiate withdrawal. It is further hypothesized that one or more proTRH-derived peptides may mediate these responses. We propose to elucidate the anatomical connectivity of this unique population of opiate-responsive proTRH neurons in the ventrolateral PAG using confocal laser microscopy and electron microscopy as a way to identify specific pathways in the brain that are involved in opiate-withdrawal responses and determine how these neurons are integrated into the control system that responds to the hyperactive state of morphine withdrawal. In addition, we will determine whether the cell specific responses of these neurons to opiate withdrawal are due to the presence of opiate receptors on these cells and if CREB or upregulation of CREB-related proteins mediate these responses. Because these neurons are in a location where they could also be involved in the modulation of pain, an increase in proTRH gene activity in the PAG following deep noxious stimulation in continuously anesthetized animals will be determined. The role of proTRH-derived peptides in opiate withdrawal and antinociception will be further studied by inhibition of proTRH gene expression using adenovirus vectors containing an antisense proTRH transgene, stereotaxically injected into the PAG and their effect on behavioral and autonomic responses during withdrawal quantified. In parallel, will identify the proTRH-derived peptides in the PAG that increase during opiate withdrawal by chromatographic and electrophoretic techniques, and based on this analysis, peptides will be synthesized and injected into discrete regions of the brain to measure its effect on withdrawal responses. As heroin addiction continues to be on the common drugs of abuse, the data generated from this proposal could have clinical significance in the design of new approaches to the treatment of addiction and the withdrawal syndrome.

This proposal involves the use of corticotrophin releasing hormone (CRH) both with and without dexamethasone for the purposes of distinguishing between Cushing's syndrome and pseudo-Cushing's in one patient. At the time of this proposal CRH was still classified as an investigational drug and, thus, could only be used under an approved compassionate use protocol. D.J. had a history of Cushing's syndrome with prior pituitary surgery. Due to her underlying depression and possible methadone interference with ACTH secretion, it was not clear whether her Cushing's had recurred. The "Dex-CRH" test was performed as previously described in the literature from researchers at the NIH to assess for this. Blood and urine tests were performed and revealed that her Cushing's had recurred.

DESCRIPTION (taken from the application) The long term goals of this study are to elucidate the mechanisms of control of the hypothalamic-pituitary-thyroid (HPT) axis under basal conditions and during conditions when this regulatory system is perturbed by environmental stressors or disease states characterized by low circulating thyroid hormone and inappropriately low TSH (nonthyroidal illness syndrome). We have recently demonstrated the presence of large concentrations of type 2 iodothyronine deiodinase (D2) mRNA in a group of specialized glial cells lining the floor and infralateral wall of the third ventricle, termed D2 tanycytes. Since D2 converts thyroxine (T4) to the more biologically active thyroid hormone, triiodothyronine (T3), we propose that D2 tanycytes comprise part of a previously unrecognized regulatory component of the HPT axis. We further propose that via interactions with the cerebrospinal fluid and neurons in the adjacent hypothalamic arcuate nucleus, D2 tanycytes modulate the set point for feedback regulation of thyroid hormone on the gene expression of TRH in the hypothalamic paraventricular nucleus. The purpose of this R21 Pilot and Feasibility Proposal is to develop animal models to test the importance of the D2 tanycyte on the regulation of the HPT axis and begin to elucidate the mechanisms by which this regulation occurs. Two different approaches to developing animal models with D2 deficiency or D2 overexpression in tanycytes will be used. In the first model, adenovirus that express antisense or overexpress sense D2 mRNA will be used to infect tanycytes after stereotaxic injection of the virus into the third ventricle or median eminence. The second model will involve the development of transgenic mice, using the DARPP-32 gene as a tool to target antisense or sense D2 transcripts to the tanycytes. Efficacy of expression of the D2 antisense and sense transcripts in tanycytes will be determined by measuring D2 activity in extracts from the mediobasal hypothalamus, and the specificity for tanycytes demonstrated by in situ hybridization histochemistry. The effect of D2 depletion and overexpression in tanycytes on the HPT axis will then be studied under basal conditions or in hypothyroid and fasting animals. Due to the coexistence of DARPP-32, a phosphatase inhibitor, and D2 in tanycytes, we will also determine whether DARPP-32 exerts a regulatory function on D2 by measuring circulating thyroid hormone levels and D2 activity in tanycytes in response to hypothyroidism and fasting in the DARPP-32 knock-out mouse.

DESCRIPTION This collaborative study will be performed in Hungary as an extension of NIH grant #RO1 DK-37021, to define the anatomical relationships between cocaine and amphetamine-regulated transcript (CART) and hypophysiotropic TRH neurons in the hypothalamic paraventricular nucleus. It is proposed that in conjunction with the parent grant, these studies will elucidate the role of CART in the regulation of hypophysiotropic TRH and determine how CART is integrated into the central control system as a mediator for the action of leptin on the hypothalamic-pituitary-thyroid axis. The origin of CART-synthesizing neurons that project to TRH neurons in the paraventricular nucleus will be identified by a two-step procedure. First, regions of the brain that contain CART-synthesizing neurons and project to the paraventricular nucleus will be identified by a double-labeling immunofluorescent technique following the stereotaxic injection of the retrogradely transported marker substance, cholera toxin subunit B (CTB), into subdivisions of the paraventricular nucleus. Second, by confocal microscopy, it will be determined whether CART-producing neurons from each of the regions where CART-IR neurons accumulate CTB, project specifically to TRH neurons in the paraventricular nucleus. This will be accomplished by a triple-labeling fluorescent technique in which the anterogradely transported marker substance, PHAL and CART will be identified by immunofluorescence in axon terminals contacting proTRH mRNA-containing neurons in the paraventricular nucleus, and proTRH mRNA will be identified by fluorescent, non-isotopic in situ hybridization histochemistry. Since CART is contained in the majority of TRH-producing neurons in the paraventricular nucleus, a double-labeling immunofluorescent technique will also be used to explore the possibility that the CART innervation to TRH neurons in the paraventricular nucleus may arise from an ultrashort feedback loop from hypophysiotropic neurons, itself. The subcellular organization of CART and TRH in these neurons will be examined by ultrastructural immunocytochemistry to determine whether both substances are packaged in the same vesicles, and whether their packaging is similarly affected by fasting. Finally, the effect of fasting and leptin administration to fasting animals on CART gene expression in hypophysitoropic TRH neurons and in CART-producing neurons that project to TRH-containing neurons in the paraventricular nucleus, will be studied using combined isotopic and non-isotopic techniques of in situ hybridization histochemistry and computerized image analysis.

DESCRIPTION (provided by applicant): Thyrotropin-releasing hormone (TRH) has an important role in the regulation of energy homeostasis not only through effects on thyroid function, but also on feeding behavior, thermogenesis, and autonomic regulation. We hypothesize that two distinct populations of TRH-producing neurons located in the medial/periventricular vs anterior parvocellular subdivision of the hypothalamic paraventricular nucleus (PVN) have critical roles in a coordinated effort to mediate the effects of TRH on energy homeostasis. To determine how these two TRH neuronal populations are integrated into energy control systems in the brain and elucidate their specific functions, we will develop transgenic mice in which Cre recombinase is selectively targeted to TRH-producing cells. This approach, together with the use of genetically modified pseudorabies virus (PRV) that exploits the Cre-loxP system for conditional replication and expression of the fluorescent marker protein, GFP, will be used to elucidate the complex, multisynaptic circuitries regulating of each of these neuronal groups and their regulation by leptin. In addition, using Cre-lox-site-specific recombination, we propose to construct a PRV derivative that is dependent on a Cre-mediated recombination event for replication, yet unable to infect other adjacent or transsynaptic neuronal populations due to a mutation in gB, a major membrane protein required for membrane fusion and transneuronal passage of virions. Thus, only neurons that express Cre will be targeted for cell death, allowing selective loss of the TRH neuronal population in each defined anatomical region of the PVN without any effects on adjacent populations. The use of these novel "suicide" viruses to selectively ablate TRH neurons in each subdivision of the PVN, will ultimately allow the elucidation of the full gambit of physiologic effects exerted by TRH neurons in the anterior vs medial/periventricular subdivision in the regulation of energy homeostasis, and may become of general value as a new neuroanatomical tool for neuroscience research.

DESCRIPTION (provided by applicant): Tanycytes are specialized ependymal cells found in high density along the floor and ventrolateral walls of the third ventricle between the rostral and caudal limits of the hypothalamic median eminence. Little is known about these cells but their expression of type 2 iodothyronine deiodinase (D2), an enzyme critical for conversion of thyroid hormone into its active form, and responsiveness to fasting and endotoxin in several animal species, has raised the possibility that tanycytes are involved in energy homeostasis and contribute to the mechanism by which circulating thyroid hormone levels fall in individuals with severe infection, termed the "nonthyroidal illness syndrome". To facilitate understanding of the importance of tanycytes in neuroendocrine regulation through effects on the hypothalamic-pituitary-thyroid (HPT) axis and feeding-related centers in the arcuate nucleus, we propose to identify tanycytes-specific genes using laser capture microdissection and microarray analysis and develop transgenic mice that express Cre recombinase specifically in tanycytes. These mice will be used to develop a tanycyte cell culture system, and to specifically ablate alpha and/or beta n vivo by developing double transgenic mice after crossing with a Cre-inducible diphtheria toxin receptor mouse line. These studies are expected to generate new information about the biologic importance of tanycytes and their role in neuroendocrine regulation of the HPT axis and in energy homeostasis. PUBLIC HEALTH RELEVANCE: Tanycytes are specialized cells that line the floor and ventrolateral walls of the third ventricle. Compelling evidence suggests they have an important role in generating thyroid hormone due to high expression of type 2 iodothyronine deiodinase, an enzyme critical for conversion of the prohormone, T4, into the biologically active thyroid hormone, T3. We have proposed, therefore, that tanycytes are involved in regulation of the hypothalamic-pituitary-thyroid (HPT) axis and energy homeostasis. To facilitate the study of these cells in neuroendocrine regulation, we will identify tanycyte specific genes by laser capture microdissection and microarray analysis to develop transgenic mice that express Cre recombinase specifically in tanycytes. These mice will be used to develop double transgenic mice in which tanycytes are selectively ablated or marked with GFP to allow isolation by fluorescence-activated cell sorting (FACS) for tissue culture.

? DESCRIPTION (provided by applicant): Senescence of POMC neurons is believed to contribute to the etiology of age-related obesity. As we have identified tanycytes to express POMC in the adult brain and recent evidence indicates that tanycytes have stem cell-like properties and can serve as progenitor cells for hypothalamic neurons, we hypothesize that tanycytes are capable of differentiating into POMC neurons in the mediobasal hypothalamus and potentially could restore failing POMC neurons in the aging brain. To begin to test this hypothesis, we propose to use the R21 exploratory mechanism to determine whether tanycytes can generate POMC neurons in the postnatal brain and integrate into the feeding-related circuitry. To perform these studies, a POMC null transgenic mouse model, ArcPomcfneo/fneoKO, which have an obese phenotype due to the loss of POMC in the mediobasal hypothalamus, will be crossed with RaxCreERT2 mice in which Cre-recombinase is expressed exclusively in tanycytes in response to tamoxifen. Therefore, all POMC neurons observed in the offspring of the resulting ArcPomcRaxRE mice can only be derived from tanycytes. We will determine whether these mice show evidence for improvement in metabolic parameters and obesity when tanycyte POMC is reactivated at postnatal day 4 vs. postnatal day 28 over a 4 month observation period. Included in the analysis will be growth, body weight, food intake, body composition, energy balance, glucose homeostasis and thyroid function. At the end of the observation period, animals will be euthanized to determine the number and location of activated POMC neurons in the mediobasal hypothalamus compared to intact and null POMC controls, and whether they migrate to their natural loci in the mediobasal hypothalamus and integrate into the feeding-related circuitry. Tanycyte-derived POMC neurons will also be phenotyped with respect to their expression of neurotransmitters and responsiveness to leptin. These studies are expected to expand the understanding of POMC system neurobiology in the hypothalamic arcuate nucleus and provide new and novel insights into ways the melanocortin system could be regulated to treat obesity and metabolic disorders associated with aging.

? DESCRIPTION (provided by applicant): We hypothesize that hypothalamic inflammation associated with high fat feeding arises as a result of Toll-like receptor (TLR) signaling in a specialized cell type lining the inferolateral walls of the third ventricle, the alpha tanycyte. We propose that the activation of NF-?B in these cells releases inflammatory mediators into feeding-related centers in the mediobasal hypothalamus, increasing feeding and/or altering the sensitivity of arcuate/ventromedial nucleus neurons to leptin and/or insulin. To test this hypothesis, RNA-Seq analysis will be used to identify the complete gene expression profile of alpha tanycytes in response to a HFD, with particular emphasis given to the identification of genes and pathways involved in inflammation. Based on this analysis, the effect of a HFD on food intake, energy expenditure, glucose homeostasis, leptin and insulin sensitivity, and other parameters of hypothalamic inflammation will be studied in transgenic animals with conditional KO in alpha tanycytes of selected genes identified in the RNA-Seq analysis. We propose that these studies will demonstrate a critical role of tanycytes in the pathophysiology of hypothalamic inflammation associated with high fat feeding, uncovering new knowledge in understanding the pathogenesis of obesity and a potentially new pharmacologic target for the treatment of obesity.

? DESCRIPTION (provided by applicant): Following fasting, satiety is observed within two hours after reintroducing food and accompanied by neuronal activation in distinct areas of the brain including the hypothalamic paraventricular nucleus and dorsomedial nucleus, parabrachial nucleus, medial and commissural subdivisions of the nucleus tractus solitarius (NTS), central nucleus of the amygdala (CeA) and bed nucleus of the stria terminalis (BNST) that have all been associated with the regulation of food intake by affecting autonomic and reward centers in the brain. In addition to these regions, we observed in our preliminary studies striking cfos activation in the parasubthalamic nucleus (PSTN), a poorly characterized nucleus in the brain that we hypothesize comprises a major node in the satiety circuitry. To test this hypothesis, we will elucidate how refeeding-activated PSTN neurons are integrated into the central nervous system using classic neuroanatomical tracing techniques (retrograde and anterograde), and to test the functionality of these neurons in the regulation of appetite, the use of DREADDs to activate or inhibit PSTN activity. Anatomical maps will be constructed of the projection pathways of refeeding-activated PSTN neurons including the topography of neurons in the PSTN that project to known appetite regulating centers and whether activated neurons have a single, dedicated projection field or multiple projection fields. The functionality of this circuitry will e tested by activating or inhibiting PSTN neuronal perikarya using AAV expressing DREADDs fused to mCherry injected bilaterally into the PSTN that either expresses hM3Dq to activate PSTN neurons or hM4Di to silence PSTN neurons in response to the systemic administration of clozapine-N-oxide (CNO). Behavioral readouts used will include measuring food and water intake and locomotor activity in automated cages using a fasting-refeeding paradigm, and by operant conditioning to study reward feeding. We propose that these studies will lead to new insights about how appetite is suppressed and better understanding about the mechanisms underlying the regulation of food intake.

Hypothalamic proopiomelanocortin (POMC) neurons are critical regulators of feeding behavior and energy balance in both rodents and humans. As tanycytes, ependymoglial cells that constitute the wall of the third ventricle adjacent to the arcuate nucleus, synthesize POMC and may function as adult, neural stem/progenitor cells, we hypothesized that they are capable of maturing into POMC neurons that populate the arcuate nucleus. To test this possibility, we created a mouse model, the RaxCreERT2/+/ArcPomcfneo/fneo mouse, in which POMC-expressing neurons would only appear in the arcuate nucleus following tamoxifen treatment if any derive from tanycytes. Our preliminary data demonstrate that ~10% of the normal population of Pomc- expressing neurons in the arcuate nucleus originate from tanycytes when tamoxifen is administered between ages 6 and 9 months, associated with partial reversal of the obese phenotype of ArcPomcfneo/fneoKO mice. To elucidate the mechanisms by which this transformation takes place, we propose to study the transcriptome by RNA-Seq analysis of tanycyte-derived, adult-born POMC neurons, singly isolated by laser capture microdissection, and compare to the rest of the arcuate nucleus POMC neuronal population and to tanycytes. This will be accomplished using a RaxCreERT2/Ai9/Pomc-EGFP mouse model in which the population of POMC neurons derived from tanycytes following tamoxifen treatment can be distinguished from all other POMC neurons and tanycytes by their fluorescence characteristics. To determine the appropriate age following tamoxifen administration to isolate POMC neurons derived from tanycytes at their earliest timepoint, initial studies will be conducted using the RaxCreERT2/+/ArcPomcfneo/fneo model and both in situ hybridization histochemistry and immunocytochemistry to determine the age of peak POMC neuronal generation and the time course by which tanycytes differentiate into POMC neurons and integrate into the feeding-related circuitry. We propose the studies could provide important, new information about POMC neurogenesis that could lead to a novel approach to treat obesity and the age-related decline of adult, hypothalamic POMC neurons.