Richard DeForest Palmiter, Ph.D. - US grants

The overall focus of this grant is to study the regulation of mouse metallothionein (MT) genes in developmental, physiological and molecular terms. We propose to isolate and characterize the mouse MT-II gene with particular emphasis on comparing its transcriptional regulation in response to various metal and steroid inducers to that of the MT-I gene. The regulatory regions of these genes will be defined by in vitro mutagenesis coupled with expression assays following injection into mouse eggs or transfection into tissue culture cells. We also propose to study the binding of purified glucocorticoid and heavy metal receptors to the regulatory regions of these MT genes. Mutations in the structural genes will be created to test: (a) the effects of introns on mRNA processing, (b) the role of untranslated regions on MRNA stability and function and (c) the importance of amino acid sequence on MT function and stability. The molecular basis of MT gene commitment will be explored by trying to define critical sites of DNA methylation that affect MT gene inducibility. Changes in MT gene commitment and expression will be correlated with changes in chromatin structure, especially with the appearance of a nuclease hypersensitive site that lies 5' of the transcription start site. The MT promoter/regulatory region will be fused to other structural genes and these fusion genes will be introduced into mice by injecting them into fertilized eggs. This system allows analysis of the effects of chromosome location on tissue specific gene expression, the inheritance of foreign genes and changes in gene expression that occur from one generation to the next. The regulation of these fusion genes by zinc permits testing the biological consequences of altering the level of expression of a particular gene product. In addition, this technique affords an opportunity to correct certain gentic defects.

Transgenic mice offer a new approach to attack complex systems such as the nervous system. A multi-faceted approach directed towards studying the consequences of interfering with neuron development and function is being developed. As first step, the four genes encoding the enzymes involved in catecholamine biosynthesis will be cloned in order to characterize the neuron- specific enhancer and promoter elements in each of these genes. To help understand the DNA elements that allow expression of these genes in subsets of neurons, the enhancers from the neuron- specific enolase gene, that is expressed in all neurons, will also be characterized. The enhancer/promoter elements from these genes will be used to direct the expression of various structural genes to specific neurons and adrenal medullary cells of transgenic mice. Structural genes will be chosen that may affect various aspects of neuronal cell development or function. They fall into several categories. (i) Oncogenes: The effect of SV40 T- antigen, ras, myc and /or src on neuronal differentiation or transformation will be studied. (ii) Nerve growth factor (NGF): The hypothesis that neuronal growth, differentiation and survival are influenced by gradients of NGF will be tested. (iii) Neurotransmitters: Transgenic mice in which specific subsets of neurons express additional neurotransmitters or neuromodulatory peptides will be created to determine whether altering neurotransmitter synthesis affects the development and function of the nervous system. (iv) Diphtheria toxin: This toxin will be used to ablate epinephrine-producing adrenal medullary cells to study the role of epinephrine in stress response. It will also be used to study the consequences of deleting neurons making a specific neurotransmitter on the development of the rest of the nervous system.

The major goals of this research are to use genetic approaches to determine: (a) whether metallothioneins (MT) are essential during mammalian development and physiology, (b) whether different isoforms of MT have distinct functions and (c) whether MTs can play a role in retarding malignant transformation. The MT-I and MT-II genes are expressed in most organs, they are inducible by a variety of metals and hormones, and they probably have a similar, general function. These MTs will be disrupted by homologous recombination in embryonic stem cells and those cells will be used to generate mice lacking MT-I, MT-II or both isoforms. The effect of these mutations on normal development and physiological responses will be examined. Mice over-expressing MT-I or MT-III (an unusual MT normally expressed only in the brain) have been generated by microinjection of marked versions of these genes flanked by presumptive locus control regions from the MT locus. These transgenic mice will be used to determine if excess or ectopic expression of MT affects development or physiology. Mice expressing reduced or elevated levels of MT will then be crossed with two different transgenic lines of mice with predispositions to develop hepatocellular carcinoma. If MT plays a role in retarding tumorigenesis, then we predict that hepatic tumors will develop more slowly in mice expressing excess MT and more rapidly in mice expressing reduced levels of MT.

environmental toxicology; biomedical facility; genetically modified animals; animal breeding; disease /disorder proneness /risk; laboratory mouse;

DESCRIPTION: This project proposes to use genetic techniques to gain a better understanding of zinc metabolism in the mouse. A family of genes encoding transmembrane proteins that facilitate removal of zinc from the cytoplasm has been cloned. This family includes ZnT-1, which transports zinc out of the cell; ZnT-2, which facilitates accumulation of zinc in endosomal/lysosomal compartments, and ZnT-3, which was cloned by homology to ZnT-2 and is expressed in neuronal cells that sequester zinc in synaptic vesicles. These genes may play an essential role in protecting cells from excess zinc and/or facilitating the concentration of zinc in intracellular compartments where it may serve special functions. To test these hypotheses, the expression of these transporters will be analyzed, at both the tissue and cellular level, and that information will be used to help interpret the phenotypes of mice in which each of these genes is inactivated by homologous recombination. Mice lacking both zinc transporters and metallothioneins will also be generated to help define the role of these metal-binding proteins. The most exciting outcome of these experiments may be the demonstration that zinc has important neuromodulator functions.

DESCRIPTION (applicant's abstract): Neuropeptide Y (NPY) has been implicated in the regulation of appetite and energy balance because (a) centrally administered NPY stimulates robust feeding, (b) NPY mRNA and protein levels rise in the arcuate nucleus of the hypothalamus under conditions of reduced energy balance, as well as in ob/ob mice that lack leptin and consequently become hyperphagic. Moreover, intervention of NPY signaling by administering anti-sense oligonucleotides, antibodies or NPY receptor antagonists generally inhibits feeding. Thus, it was a surprise that knock-out mice unable to make NPY had normal body weight regulation. The genetic results clearly indicate that NPY is not essential for feeding under the conditions examined, but they do not address the question of whether NPY is acutely involved in regulation of appetite. It is possible that chronic absence of NPY triggers compensatory mechanisms. This proposal aims to use genetic techniques to explore several potential forms of compensation. The specific aims address the following questions: (1) Does agouti related protein (AgRP) compensate for NPY deficiency?, (2) Do the neurons that make NPY and AgRP in the arcuate nucleus produce other neuromodulators that may compensate for NPY deficiency?, and (3) Does acute inactivation of NPY gene expression in the adult affect appetite and energy balance? The first aim will be addressed by generating mice in which both NPY and AgRP genes are inactivated. If AgRP compensates for NPY, then these mice should be lean. The second aim relies on genetic ablation of the neurons that make NPY and AgRP. If those neurons are important for energy balance, then those mice should be lean. The last aim is addressed by creating mice with a NPY gene that can be inactivated at will. Inactivation of NPY expression in the adult may have transient or chronic effects on regulation of appetite and energy balance. These genetic experiments should help rationalize the current disparate results obtained by permanent NPY gene silencing and by acute intervention of NPY signaling.

DESCRIPTION (provided by applicant): A small population of neurons (~ 5000 cells) in the arcuate region of hypothalamus that make neuropeptide Y (NPY) and agouti-related protein (AgRP) become essential for survival in adult mice. We targeted the diphtheria toxin receptor (DTR) to these NPY/AgRP neurons to allow their ablation by administration of diphtheria toxin (DT). Ablation of these neurons in adult mice results in starvation. However, their ablation in neonatal mice results in circuit-based compensation such that mice grow almost normally. Although neonatally DT-lesioned mice grow to normal size, we experimentally explore whether they manifest deficits in physiological responses that normally depend on NPY/AgRP neurons. We propose to determine the critical signaling molecules made by NPY/AgRP neurons and whether they function primarily by regulating the melanocortin-signaling pathway or other circuits. A prime candidate for the critical signaling molecule is ?-aminobutyric acid (GABA);thus, we propose to make conditional alleles of the two genes, GAD1 and GAD2 encoding the biosynthetic enzymes such that they can be inactivated in NPY/AgRP neurons and temporally controlled manner by the action of Cre recombinase. If GABA is the critical signaling molecule, then we predict that inactivation of GABA biosynthesis in NPY/AgRP neurons of neonatal mice will lead to compensation such that the cells are no longer necessary for survival. In contrast, inactivation of GABA biosynthesis in NPY/AgRP neurons of adult mice may promote starvation. NPY/AgRP neurons are known to inhibit neighboring neurons in the arcuate nucleus that make proopiomelanocortin (POMC) and their target neurons in the paraventricular nucleus that express melanocortin-4 receptor. Activation of POMC neurons inhibits feeding. Thus, we include experiments to directly test whether up-regulation of this melanocortin-signaling pathway after sudden ablation of NPY/AgRP neurons is critically involved in the starvation phenotype. However, because NPY/AgRP neurons also project to several other brain regions, we include experiments that address which of these projection regions are affected the most and whether they contribute to feeding behavior. These experiments should provide insight into the neural circuits and signaling molecules that are critical for feeding.

The goal of this project is to use a combination of genefically engineered mice and virus-mediated gene transfer in conjuncfion with mouse behavioral tests of learning and memory to identify the neural circuits that may underlie the cognifive decline in PD pafients. We will develop two novel transgenic mouse lines that will allow us either to block genefically the producfion of dopamine in discrete dopaminergic projecfion regions by viral-mediated recombination of the tyrosine hydroxylase gene or to ablate completely dopamine neurons. We will determine whether the loss of dopamine signaling (by inacfivafion of tyrosine hydroxylase) or dopamine neuron death (by action pf diphtheria toxin) leads to cognitive impairment and morphological changes within the striatum and/or prefrontal cortex (in conjuncfion with project 2). RELEVANCE (See instructions): Parkinson's disease (PD) is caused by dopamine neuron cell death, but it is unclear if PD-related cognifive impairment is due to the loss of dopamine signaling, or to the secondary effects of dopamine neuron degenerafion. We will develop 2 novel mouse models of PD to distinguish between the effects of loss of dopamine signaling and dopamine neuron degeneration on cognifive abilities.

Abstract/Summary The loss of appetite (anorexia) associated with eating too much, food poisoning, and nausea is mediated by a neural circuit that starts with activation of vagal afferents, which activate brain stem nuclei and then neurons that express calcitonin-gene-related protein (CGRP) in the parabrachial nucleus (PBN). Transient activation of these CGRPPBN neurons by chemogenetic or optogenetic means is sufficient to inhibit feeding and to induce conditioned taste aversion, the phenomenon by which animals learn to avoid foods that make them sick. Chronic activation of CGRPPBN neurons by various genetic manipulations can result in starvation. Chronic activation of these neurons, as occurs during illnesses such as cancer, promotes anorexia. The CGRPPBN neurons project their axons to forebrain structures, including the central nucleus of the amygdala (CeA). Photoactivation of CGRPPBN axon terminals expressing channelrhodopsin in the CeA also inhibits feeding. This grant proposes to establish the molecular identity of the neurons in the CeA (and possibly other brain regions) that mediate anorexia and then extend the neural circuit by molecular characterization of the neurons that are downstream of the CeA neurons. Thus, this research will extend the ?anorexia neural circuit? by at least two nodes. The molecular profile will be determined by quantification of all mRNAs being translated by the neurons at each of these nodes. That information will provide valuable clues for extending the neural circuit and devising therapeutic solutions to anorexia caused by chronic illness. The results of these experiments will also provide a blueprint for understanding how memories of food-related illness and nausea are established.

Project Summary Learning to avoid environmental threats is essential for survival. Our knowledge of the neural substrates within the central nervous system responsible for learning to avoid such threats is incomplete. The precise analysis of the function and regulation of specific neurons within neural circuits that mediate threat perception and avoidance is essential for our understanding of these basic neural processes and the etiology of neurological and psychiatric disease. We have identified a population of neurons within the parabrachial nucleus (PBN) that express calcitonin gene-related peptide (CGRP). This specific population of neurons is necessary and sufficient for mediating behavioral responses to foot shock and visceral malaise. Experimental activation of CGRP-neuron terminals in the central nucleus of the amygdala is sufficient to generate fear or taste memories; however, activation of CGRP receptor (CALCRL) neurons in the CeA is sufficient for fear conditioning but not for taste conditioning. This multi-investigator proposal will draw on the expertise of three PIs (Dr. Zweifel, Dr. Palmiter, and Dr. McKnight) to discover how different threats are differentially recognized by the central nucleus of the amygdala (CeA). To address this fundamental question we will integrate cutting-edge techniques in mouse genetics, viral-mediated circuit dissection, behavior, in vivo imaging, electrophysiology, and cell-specific molecular profiling. We will characterize the molecular profile (translated mRNAs) of postsynaptic CGRP receptor (CALCRL)-expressing and non-CALCRL expressing neurons in the CeA to establish the identity of these neurons recruited during conditioned taste aversion and fear. We will elucidate the extent of activation of these circuit components using in vivo calcium imaging of circuit dynamics in freely behaving mice. We will establish how distinct noxious stimuli associated with pain or visceral malaise diverge at the level of CALCRL-and non-CALCRL expressing neurons in the CeA through functional manipulation of these neural circuit connections with light- and drug-activated receptors. Successful completion of this proposal will delineate key circuit components underlying aversive processing in the brain and will serve as a gateway to future investigations into downstream circuit components critical for these processes.