Jeffrey M. Friedman - US grants

Over the last 50 years, the mouse have proven to be a very useful organism for the study of mammalian physiology and disease. Genetic studies in mice have defined numerous instances where either single genes or multiple genes control the inheritance of a particular phenotype. The phenotypes available for study in mice span a broad range and include such diverse biologic problems as obesity, hypertension, hyperlipidemia, cancer, diabetes, behavior and development. The specific aims of this proposal are to generate a genetic map in mice using DNA markers with RFLPs spaced at approximate 10 cm intervals. The DNA probes to be used in these mapping experiments have been chosen so as to generate a map of particularly useful markers and include: 1) VNTR clones, which are highly polymorphic, 2) Not 1 containing clones, which are useful for physical mapping as well as chromosome hopping, 3) anchor loci known to be on each mouse chromosome. The availability of a genetic map composed of these markers will be useful for the genetic analysis of the more than 1300 mouse mutations, the analysis of polygenic inheritance and may also expedite efforts to physically map the mouse genome.

Obesity is a major source of medical morbidity (hypertension, diabetes, hyperlipidemia); in the U.S., approximately 25% of adults and 20% of children are obese. Although this disorder must ultimately be due to an imbalance in food intake and energy expenditure, intensive clinical and animal investigation has failed to elucidate the molecular-physiologic mechanism for obesity in any single instance. Mice homozygous for either the ob or db gene display a metabolic phenotype which closely resembles that of the obese human. Animals homozygous for either mutation, whose genes lie on different chromosomes, are massively obese and frequently weight 3 times as much as lean littermates by 10 months of age. These animals also demonstrate: enhanced metabolic efficiency, hyperphagia, peripheral insulin resistance and diabetes mellitus. Many attempts have been made to identify the specific protein or pathway responsible for the phenotype of these animals. However, it is unclear which differences (if any) are primary (i.e. directly related to the gene) and which are secondary to the profound metabolic alterations it produces. This very inability to disentangle primary from secondary events is the most compelling argument for the use of reverse genetics in delineation of the specific lesion in these animal models. The ob and db genes, which appear to control separate aspects of a single metabolic pathway or control loop related to energy homeostasis will be genetically mapped, and the gene having most closely linked RFLPs selected for molecular cloning and characterization. Employing techniques of "reverse genetics", the ob and db genes will first be mapped genetically on large backcrosses relative to appropriate genomic probes, and then isolated by physical methods including pulsed field gen electrophoresis, cosmid clones and/or yeast artificial chromosomes. Gene identity will be assessed by screening for tissue- specific expression of candidate sequences, and their subsequent "complementation" of the mutant phenotype of transgenic insertion. Our proximate aim is the localization and preliminary characterization of the genomic lesions in one of these forms of mouse obesity. Identification of the genomic sequences (or closely linked RFLPs) causally related to these animal obesities may permit direct mapping of these genes on relevant human pedigrees. Such linkage analysis would permit rapid assessment of the relevance of any mouse genomic sequence to obesity in man. Most importantly, the basis for at least one type of genetic obesity will be understood at the level of gene action.

The assimilation, storage and disposition of nutrient energy constitute a complex homeostatic system central to the survival of metazoa. In vertebrates, and particularly among land dwelling mammalian species, the ability to store large quantities of chemical energy in the form of adipose tissue triglycerides is crucial for surviving long periods of food deprivation. In order to maintain such food stores without sustaining continual alterations in the size and shape of the organism, a balance between energy intake and expenditure must be achieved. Despite intensive investigation, the molecular mechanisms which regulate energy intake and energy expenditure remain to be elucidated. It is anticipated that the identification of molecules that transduce nutritional information and regulate these functions will be critical to our understanding of the regulation of body weight in health as well as in disease states such as cancer. It is the objective of this application to isolate two of the genes which regulate this system: the mutant mouse genes obese (ob) and diabetes (db). These recessive mutations cause a severe obese phenotype which resembles human obesity in a number of critical aspects. The genes will be cloned using the techniques of positional cloning. RFLPs which flank these mutations are currently being used to initiate chromosome walks using yeast artificial chromosomes to isolate the ob and db genes. Candidate genes which have been genetically localized to the regions of ob and db will be tested for their ability to complement the mutations when introduced into transgenic or ES cell-derived mice. Once the genes have been isolated, a series of experiments will be initiated to determine the mechanism(s) whereby these genes control body weight. These studies will include a computer analysis of the predicted protein sequence, analysis of the sites of synthesis of the encoded RNA and protein, isolation of the human homologues of ob and db analysis of the regulation of the genes under physiologic and pathophysiologic circumstances, and functional studies of the encoded proteins in vitro and in vivo.

technology /technique development; male; female; nutrition; human subject; genetics; hormones; endocrine gland /system; biomedical resource; biological products; Mammalia;

[unreadable] DESCRIPTION (provided by applicant): This application proposes that experiments that will contribute to our understanding of the causes of obesity and Type 2 diabetes and could improve our ability to treat these conditions. Toward that end we propose to establish the precise sites of action of the hormone leptin and the molecular mechanisms that account for its novel metabolic effects. Leptin is a hormone that is produced by adipose tissue and sends nutritional signals to a number of other tissues including brain. Leptin functions as an afferent signal in a feedback loop that maintains constancy of the total body fat mass. A mutation in the leptin gene in ob/ob mice or some human subjects leads to severe obesity. In most cases, obesity is the result of a relative insensitivity to the effects of this hormone. In leptin sensitive animals, this hormone exerts a number of novel metabolic effects. It reduces the cellular levels of lipid and improves insulin action. Leptin is also capable of correcting the severe diabetes that develops in human subjects with lipodystrophy (the absence of fat tissue). This effect is the result at least in part, of the ability of leptin to reduce lipid content in liver and other tissues. In recent work we have shown that leptin's effects on cellular lipid content and body weight is the result of its ability to repress SCD1, an enzyme that converts saturated fatty acids to mono-unsaturated fatty acids. In this application, we propose to follow up on these results by 1) using modern methods in genetics to study the effects of selective ablation of the receptor for leptin in a variety of tissues, 2) study the mechanism by which repression of SCD1 reduces body weight and cellular lipid content 3) assessing the role of other gene products in mediating the biologic response to leptin 4) studying the mechanism by which leptin regulates SCD1 and other genes. This latter effort will make use of biologic and computational approaches. In aggregate, these studies will add to our understanding of the molecular mechanisms regulating body weight and metabolism and potentially lead to new therapeutic approaches. [unreadable] [unreadable]

The nervous system poses unique challenges to gene expression analysis because of its extreme cellular heterogeneity and complex distributions of messenger RNAs within individual cells. Indeed the number of neural phenotypes is largely unknown. We propose to develop new methods for analyzing neural phenotypes by using cDNA from neurons that are fluorescently labeled in vivo as targets for hybridization to microarrays. These methods will require the coordinate development of new hardware and software tools. Toward that end, a collaborative program among molecular biologists and computational biologists is proposed that will: 1) develop new hardware for microarrays by comparing the performance of Gel pad microarrays, provided as part of collaborative studies with the Motorola Corporation, to alternative methodologies. Gel pad microarrays represent a new technology that has several theoretical advantages relative to glass microarrays or arrays of oligonucleotides 2) develop methods for the preparation of cDNA from single neurons or at least single neuronal types. cDNA will be prepared from neurons that have been marked in vivo in transgenic mice in which the expression of fluorescent proteins has been using modified BACs. Different "color" fluorescent proteins are first being to neurons expressing NPY (Cyan Fluorescent protein) or POMC (Topaz Fluorescent protein) 3) develop new for tracking, management, querying and retrieval of generated expression data and identification of expression data points within the collected images. In addition, new algorithms for clustering cell by analyzing the level of expression of a neuronal RNAs to those in a pool of CY3 labeled RNA from ten mouse organs. These algorithms will also incorporate data from a set of 100 unchanging RNAs identified and independently quantitated (Specific aim 3). It is widely believed that the use of cDNA microarrays will have an enormous impact on biological research. This impact is likely to be especially great in efforts to study the nervous system where even the total number of cell types is not entirely clear. The methods proposed in this are designed to advance this technology to establish the phenotype of neural cells.

DESCRIPTION (Adapted from investigator's abstract): This application proposes a series of genetic and clinical studies to elucidate the genetic basis of hypertension, obesity, diabetes and dyslipidemia. These diseases are often associated with one another and when they occur together they are referred to as Syndrome X. The basis for these disorders, which are principal causes of morbidity and mortality worldwide, is largely unknown. The incidence of obesity, hypertension, diabetes and dyslipidemia is increased on the Pacific Island of Kosrae. In studies completed in 1994, a medical evaluation was conducted and DNA was isolated from each of 2364 Kosraeans over the age 20, nearly the entire adult population. In this application, the investigators propose to complete this genetic study by applying modern methods in human genetics to map the genes that cause the components of Syndrome X. In addition, a follow-up clinical study will be performed on the entire population of the island over 16 years old. It is expected that this will add 1000 new participants.

leptin; biofeedback; weight loss; human therapy evaluation; obesity; reducing diet; basal metabolism; hormone therapy; caloric dietary content; adipose tissue; metabolism disorder; endocrinology; endocrine disorder; patient oriented research; human subject; clinical research;

DESCRIPTION (provided by applicant): This proposal outlines the development of novel methods for the mapping of complex neural pathways and the specification of the neurons that compose these pathways. Our groups have recently made a recombinant Pseudorabies virus (PRV) that is conditional on cre-recombinase for viability and expression of a GFP reporter gene. This virus has been used successfully to map inputs to specific neuronal populations in defined brain regions. For simplicity we will refer to this and related methods as PAM, Pseudorabies virus assisted mapping. In this application, we propose to further develop PAM so as to allow: 1) Simultaneous Retrograde Tracing from Different Neurons in the Same Brain Region, 2) Simultaneous Retrograde Tracing from Similar Neurons in Different Brain Regions, 3) Anterograde tracing 4) Identification of Neurons Projecting to More than One Site 5) Specification of the neurons that are identified in these mapping studies. To establish the validity of the approach, these studies will be performed in analyses of hypothalamic circuits that regulate feeding. Once the methods are validated, the viruses and animals will be made freely available to other investigators interested in analogous studies of other neuronal pathways.

DESCRIPTION (provided by applicant): Leptin is an adipocyte hormone that functions as the afferent signal in a negative feedback loop that regulates adipose tissue mass. As a key element of this feedback loop, leptin both responds to changes in adipose tissue mass and in turn modulates the size of the adipose depot. This application proposes a set of experiments that seek to elucidate the molecular mechanisms that are responsible for this reciprocal relationship between leptin and adipose tissue mass. In the first set of experiments, we propose elucidate the molecular mechanisms that control leptin gene expression. Leptin plasma and RNA levels can vary several hundred fold between the fasted and obese states and the transcriptional mechanisms responsible for its adipose tissue specific expression and this quantitative regulation are not known. Leptin is expressed at significantly higher levels in vivo vs. in vitro making it necessary to study leptin gene expression using transgenic mice. We have used an in vivo luciferase imaging system to localize the cis elements necessary for tissue specific and quantitative regulation to between -22 kB and +18 kB of the leptin gene. We propose further experiments to test a set of promoter deletions to map the cis elements and trans factors regulating leptin expression as a prelude to defining the relevant signal transduction pathway. We hypothesize that leptin is regulated by a lipid sensing system and, if true, these experiments could lead us to understand how intracellular lipid content is sensed and read out by the leptin gene, In the second set of experiments, we will explore the mechanism by which changing leptin levels control adipose tissue mass. While leptin deficient obese mice show a massive increase in the number of fat cells, the factors regulating fat cell production are largely unknown. We have recently identified an adipocyte stem cell that is capable of reconstituting a fat depot and correcting the metabolic abnormalities of fat deficient lipodystrophic mice. We now propose to further characterize this cell type in vivo and in vitro as a prelude to studies of the effects of leptin on the growth and development of this novel cell type. We also propose to study adipose tissue development by employing an ES cell complementation method that we have developed. In this method, wild type ES cells are injected into blastocysts of lipodystrophic animals. In the resulting chimaeras, the ES cells are the sole source of adipose tissue. This technique will allow us to titrate the number of ES cells to define the minimal clone size required for development of the adipose mass, results which will have important implications for our understanding of the ability of adipose tissue precursors to reconstitute adipose tissue. This method also provides a robust and efficient means for studying the role of specific gene products that will be identified in the other experiments in regulating adipose tissue development and function. PUBLIC HEALTH RELEVANCE: Obesity is associated with Type II diabetes, hypertension, hyperlipidemia and hepatic steatosis and represents a major public health problem (1). Leptin, an adipocyte hormone, regulates adipose tissue mass as part of a feedback loop and a fuller understanding of the elements of this system could have important implications for the pathophysiology and treatment of obesity. In this application, we propose to address two unanswered questions in leptin physiology. What controls the amount of leptin that is produced in the lean vs. obese state? How do changes in leptin concentration in turn regulate adipose tissue mass?

DESCRIPTION (provided by applicant): A set of experiments is proposed to validate and further develop a new nanoparticle based technology, Nanoparticle induced Circuit excitation (NICE), for modulating the activity of cells remotely and non-invasively. A fundamental goal of biology is to understand the role of each cell type in a complex organism. The definitive test of cell function is to selectively turn on or off the activity of a single cell type in a living animal and examine the effect on physiological function. Recent tools, such as light activated ion channels such as channel rhodopsin, have pioneered the external control of membrane potential in genetically defined cells and established a new means for investigation by neuroscientists. However, these optical methods have practical disadvantages limiting their application including the need for surgical implantation of invasive fiber optics;the inability to stimulate cells in multiple anatomical regions simultaneously;and the difficulty of modulating multiple cell types in parallel. We address this challenge by using nanoparticles to activate defined cell populations remotely with radiowaves. Ferrous oxide coated with streptavidin is used to decorate cells, which express a biotin acceptor protein under the control of cell specific promoters. These same cells are engineered to also express TRPV1, a single component, temperature-sensitive ion channel that can detect small changes in temperature within the physiological range and by conformational change allow graded calcium entry. Exposing the metal coated cells to a defined electromagnetic field increases the local temperature and activates TRPV1 channels resulting in a Ca2+ current and cell activation. We have preliminary data that confirms the efficacy of this method in vitro and now propose to extend our studies to further validate the technology in vitro and to modulate in vivo functions such as hormone release and neural activity. We will also establish a means for combinatorial activation of different cells using a modified TRPV1 and nanoparticles fabricated from other metals that can be excited at different wavelengths. We will use this tool to examine the roles of specific peripheral and CNS cell populations in energy metabolism. We propose to develop this method in three stages: 1) Validate the safety and utility of NICE in vitro and refine the methodology by decorating different cell types with distinct particles tuned to different wavelengths to activate ensembles of different cell populations in various combinations. 2) Establish the ability of NICE to modify hormone release to regulate glucose metabolism in diabetic animals in vivo. 3) Show that NICE can be used to stimulate action potentials in electrically excitable cells to modify behavior and use NICE to investigate the role of specific hypothalamic populations in (NPY and POMC) to control appetite. In time, NICE may be adapted for clinical uses, e.g: induced pluripotent stem cells engineered to express NICE constructs may act as autografts to enable external control of cell function. These applications are distant but not inconceivable and the studies proposed may form the foundation for the clinical use of nanoparticles. PUBLIC HEALTH RELEVANCE: This proposal aims to validate a novel technology, Nanoparticle Induced Circuit Excitation (NICE), for external, non-invasive activation of defined cell populations in living animals. We propose to apply this methodology to express insulin and thus control glucose in diabetic animals and to modulate feeding behavior by activating specific neural populations. The studies proposed in this application will thus validate the use of NICE and provide a foundation for the eventual use of nanoparticles in clinical settings.

Project Summary In this collaborative and interdisciplinary application, we propose to develop further a novel non-invasive method for cell regulation (NICR) that is suitable for preclinical proof of concept studies. This technology potentially could be used to treat neurologic diseases and provide a less invasive alternative to deep brain stimulation (DBS) or optogenetics. We thus propose to refine the technology and develop a prototype device to test the use of NICR for the treatment of symptoms of Parkinson's Disease (PD) in mice. Cell activity is controlled by two components; the iron binding ferritin protein that spontaneously forms 5 nm iron nanoparticles and TRPV1, a temperature and mechano-sensitive channel. By tethering ferritin to TRPV1, one can gate the channel with radiofrequency (RF) (which heat or induce mechanical motion of ferritin) or a magnet (which induces motion). The method has been shown to be capable of controlling neural activity in vitro and in vivo, the latter by increasing neural firing. In addition, we have introduced a mutation into TRPV1 that converts it into a chloride channel, and the use of the mutant channel makes it possible to inhibit neural activity using electromagnetic waves (e.g., RF). Because the system is genetically encoded, one can regulate the activity of cells into which the two protein components of the system have been delivered by recombinant Adeno- Associated Virus (AAV) strains. AAV has been used in numerous human studies including patients with PD. Thus NICR could provide a less invasive alternative to implanted electrodes (DBS) or implanted light devices (optogenetics) for the modulation of neural activity (deep brain stimulation) and also be used to simultaneously control several different nodes in a neural circuit. In this application, we propose a set of preclinical proof-of-concept studies for the treatment of PD including: 1) refinement of the technology to improve its efficiency and to create suitable AAV strains to ameliorate the symptoms of PD. We also propose to increase the sensitivity of the system by using channels that can be gated with lower field strength and by identifying variants of ferritin with enhanced sensitivity to an electromagnetic field; 2) development of a prototype device that would create local electromagnetic fields of suitable strength with the aim of enabling the use of the method in routine laboratory settings and ultimately as a portable/wearable device; 3) testing the ability of the improved method and suitable instrumentation to alleviate the symptoms of PD in mice; and 4) creating knockin mice with cre dependent expression of the constructs to assess the safety of long term TRPV1 and ferritin expression. The validation of this technology could also lead to its use for the treatment of other diseases at sites within and outside the nervous system to either increase or decrease cell activity or regulate protein production. Finally, the further development of NICR could impact basic research by allowing the non-invasive activation or inhibition of cells by simply mating genetically modified mice and exposing them to RF or magnetic fields.