Dona Chikaraishi - US grants

Tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, is expressed in adrenergic neurons and the adrenal medulla. We propose to study three systems in which TH is regulated by external factors. The first is glucocorticoid (dexamethasone) induction TH enzyme activity in clonal pheochromocytoma cell lines (PC12 and derivatives). This induction appears to be typical of steroid-induced transcription such that the steady state concentration of TH RNA is increased 3-10X. We proposed to determine a) if rate of TH transcription is increased (by assaying TH "run-off" RNA in isolated nuclei); b) to identify flanking regulatory DNA sequences which bind the glucocorticoid receptor; c) to assay the biological function of such sequences by transfection of hormone-responsive transcription. The second project concerns how TH is induced in vivo by neuronal activity in sympathetic neurons and in the adrenal medulla. We plan to determine if increased trans-synaptic activity, mediated by the nicotinic acetylcholine receptor, increases TH transcription by measuring both steady state RNA levels and the rate of TH transcription. In addition we want to develop culture conditions in which trans-synaptic induction can be achieved in cell lines. Thirdly, we want to ask if the transmitter switch from adrenergic to cholinergic in primary sympathetic neurons, induced by non-neuronal cells or media from such cells, reflects changes in gene expression. Using TH probes, we will determine the nuclear and cytoplasmic TH RNA levels in adrenergic vs. cholinergic cultures. In addition, we want to clone the gene for choline acetyltransferase (CAT) the enzyme responsible for acetylcholine synthesis, since its activity is induced 50-1000X during the phenotypic switch. In addition, we propose to delineate the organization of the TH gene with regard to promoter, exon/intron and termination sites.

We and others have shown that there may be as many as 150,000 different mRNAs expressed in the rodent brain, most of which are brain-specific, arise after birth and are present at very low concentrations when whole brain is examined. Based on RNA complexity and analysis of clonal neural cell lines and brain sections, most of these mRNAs result from the composite nature of the brain, and any given cell requires only a subset of the entire brain repertoire. Surprisingly, half these mRNAs are not polyadenylated at their 3' termini, unlike most eukaryotic RNAs. We have begun to clone DNA sequences encoding some of these rare transcripts and would like to expand this array to include more that are 1) brain-specific; 2) developmentally regulated; 3) poly A-; and 4) cell-type specific; i.e. expressed in certain neural cell types or in specific regions of the brain such as the olfactory epithelium. By hybridization to the appropriate cellular RNAs, we can determine if such clones are post-transcriptionally regulated or modulated by environmental factors. Interesting clones that encode polysomal RNAs will be sequenced and the predicted peptides synthesized to generate antiserum for use in immunohistochemistry in brain sections as well as for biochemical identification of the putative proteins. In addition, direct in situ hybridization will be attempted. Southern blot analysis and isolation of longer genomic clones (from a Lambda library) should allow us to assess whether such clones rearrange during development, are members of brain-specific multi-gene families or exhibit unusual properties, such as being intronless, as has been suggested for the poly A- mRNAs. Using the same cloning approaches, we would like to study a simpler neuronal system, the olfactory epithelium. Basal cells in the epithelium continually divide to replace old olfactory neurons, and division can be synchronously induced by cutting the axons of the receptor neurons which project back to the olfactory bulb in the brain. We propose to prepare libraries (both cDNA and rare transcript-genomic libraries) to RNA extracted from rat olfactory epithelium to select clones that are specific to the receptor cells and those that are specific for the dividing stem cell.

Very few neuronal cell lines exhibit differentiated properties of mature neurons. Most existing lines are immature and neuroblast-like, which limits their usefulness. Often the identity of the parental cell type from which the lines arose is unknown. We propose to derive cultured cell lines of catecholaminergic neurons or adrenal medullary cells from tissues of transgenic (TG) mice in which oncogenes are expressed from the rat tyrosine hydroxylase (TH) promoter. Initially we will determine which regulatory elements are necessary for cell specific expression of tyrosine hydroxylase in transgenic mice using both oncogene and non-oncogene reporter genes. We are particularly interested in determining if different TH expressing cells (CNS vs PNS or dopaminergic vs noradrenergic CNS neurons) require different enhancer elements. Transgenic animals will be generated with the appropriate TH enhancer-oncogene constructs and catecholaminergic tissues from oncogene-expressing animals will be cultured. Cell lines will be selected and characterized for a variety of neuronal specific traits. If necessary animals bearing several different oncogenes will be breed to increase the efficiency of immortalization. Lastly, TH+ cells will be ablated during development by targeting the expression of the diphtheria toxin gene or the Herpes simplex virus thymidine kinase gene to catecholaminergic cells in transgenic mice.

bioimaging /biomedical imaging; imaging /visualization /scanning; technology /technique development

[unreadable] DESCRIPTION (provided by applicant): This is a renewal application for years 33-37 of GM07171 which supports the Medical Scientist Training Program (MSTP) at Duke University. One of the first MSTP's, this Program has been continually funded by the National Institutes of General Medical Sciences, the first ten years as GM01678 (1966-1975) and the subsequent 29 years as GM07171 (1975-2004). The Program currently has an award of 36 funded positions and is requesting the same number of slots in this renewal. However, the Program has grown significantly since the previous application in 1996 when there were 42 students. Currently, there are 73 students and the Program is now at a new steady state of 70-84 students. This number represents 10 to12% of the Medical School class. The additional funding is provided by the Medical and Graduate Schools, the Duke Medical Alumni Association, and the Duke Endowment. In addition, a $1,000,000 reserve fund has been created to cover unexpected financial needs by the Program. The current Program student body is quite diverse with 19% underrepresented minority enrollment and a gender distribution that is 45% female. A large pool of applicants (200-300 per year) has allowed us to recruit an outstanding group of students. The structure of the Program is unchanged since the prior application. It consists of a basic science core year, a clinical core year, graduate school, and return to a final clinical year. The average program length is 7.4 years. Students may spend their graduate years in the traditional Medical School basic sciences, Chemistry, Zoology, and Biomedical Engineering. Greater flexibility is now available to our students who may also opt a new Biostatistics and Bioinformatics Department, Economics, or Cognitive Neuroscience/Psychology. [unreadable] [unreadable] [unreadable]