James L. Roberts - US grants

The proopiomelanocortin (pro ACTH/endorphin, POMC) peptide hormone system is of central importance in animal physiology in that it has a variety of endocrine functions as well as central nervous system functions. Because of the different functions of the POMC derived peptides, the expression of these peptide hormones is regulated in a complex fashion. In this research program, it is our aim to understand how the POMC gene is regulated in the many different cell types which express it. Specifically, we wish to examine in more detail the regulation of the POMC gene in the pituitary, building on our previous work. Using POMC cDNAs and genomic DNAs, we plan to identify at which levels (transcription, RNA processing, translation, or RNA stability) glucocorticoids, estrogens, corticotropin releasing factor, dopamine and norepinephrine act to control POMC production in the pituitary. In addition, POMC gene expression in the hypothalamus will be investigated using molecular biological methods which have been developed to deal with the extremely small number of POMC producing neurons in this tissue. using the Palkovits punch technique to isolate discrete hypothalamic regions and in situ hybridization histochemistry to study POMC mRNA in individual cells, we plan to identify those factors (steroid hormones, neurotransmitters, etc. as well as physiological stimuli such as stress) which modulate POMC mRNA levels and hopefully elucidate the mechanism of that modulation. Using similar techniques as well as organ culture the developmental pattern of POMC gene expression in the fetal rat will be studied. These studies will lead to a better understanding of how an important endocrine and neromodulatory peptide hormone system is regulated at the molecular level.

The overall objective of this research program is to elucidate the molecular mechanisms involved in both the nucleus and the cytoplasm which are responsible for the alterations in mRNA levels of three important reproductive hormones, proGnRH, beta- LH and beta-FSH. An interdisciplinary approach will be taken using the tools of molecular genetics and histology in a well characterized rodent reproductive model system, that of castration and estrogen/androgen replacement in female and male. There are two aspects to the proposal which can be divided upon anatomical lines. The first is the study of gonadotropin releasing hormone (GnRH) gene expression in the preoptic area of the rat. Studies will be performed using in situ hybridization techniques, solution hybridization/nuclease protection techniques and transcription run on assays to determine how the proGnRH gene is regulated at the molecular level in this model system. In addition, the proGnRH gene promoter will be fused to a chloramphenocol acetyl-transferase reporter gene and used in subsequent gene transfection studies into various types of cultured cells to determine which second messenger systems and/or steroids are responsible for regulation of proGnRH gene expression. In situ hybridization studies in the proGnRH expressing region of the preoptic area of the rat will be performed using exon and intron specific probes to determine levels of cytoplasmic mRNA and nuclear hnRNA gene expression, respectively. Secondly, studies will also be performed in the pituitary to analyze the molecular mechanisms of beta-FSH gene expression using the castration with steroid replacement model in male and female rats as well as in primary cultures of anterior pituitary cells. Studies will be performed in primary cultures using GnRH/GAP/inhibin/sex steroids to determine which of these factors are responsible for the molecular mechanisms of regulation of beta-FSH gene expression. Analyses will be performed by transcription run on assays as well as solution hybridization/nuclease protection assays using intron/exon junctional probes developed for the beta-FSH gene in rat. In situ hybridization studies will also be performed on beta-FSH and beta-LH using mRNA and intron specific probes in the castration plus estrogen/androgen replacement paradigm in male and female using pituitary gland sections. Finally, studies on beta-LH gene expression will be extended to analyze the molecular mechanism by which the non-transcriptional effects of GnRH elicits increases in beta-LH gene expression. If regulation of beta-FSH gene expression is also found to be non-transcriptional, similar studies will be performed for beta-FSH. These studies will focus upon electron microscopic in situ hybridization studies analyzing the subnuclear level of expression of beta-LH hnRNA as well as studies on regulation of polyadenylation of beta-LH hnRNA. These studies should provide a more detailed understanding the basic molecular mechanisms involved in the regulation of these extremely important reproductive peptide/protein hormones.

The goal of this program is to elucidate and characterize the cellular and molecular mechanisms responsible for the CRH mediated activation of POMC gene expression in the anterior pituitary corticotroph. This proposal is supported by our and others previous work showing the Importance of calcium as well as cAMP in mediating CRH signal transduction and our recent Identification of novel cis-acting elements in the POMC promoter (-700/+63) responding to CRH. These studies will be performed in the well established AtT20 tumor cell system. The AtT20 cell line has been utilized extensively for investigating POMC gene expression and Is Ideal for the proposed biochemical experiments. The first aim concerns elucidating the exact roles of calcium and cAMP In mediating CRH-stimulated signal transduction resulting in transcriptional activation in the nucleus. Intracellular calcium will be quantitated by microfluorescence measurements and compared to levels of POMC transcription measured by primary transcript solution hybridization/nuclease protection and run-on transcription assays. Two basic questions will be addressed. a) Is basal intracellular Ca++ sufficient of is an elevation required for CRH/cAMP stimulated POMC and c-fos gene expression? b) What is the mechanism of Cd++ inhibition of basal and CRH activated POMC transcription and is c-fos involved? The second aim will characterize in detail the major cis-acting elements (promoter region 234/-133) responsible for mediating CRH activation as well as the transacting factors regulated by the signal transduction events investigated in the first part of this program. Four issues will be addressed. a) Determination of the basal and CRH induced factors (c-fos?) which interact with the major (-234/-133) CRH regulatory POMC promoter element. b) Functionally dissect the -234/133 region with specific promoter mutants to determine the relative contribution between basal and regulated cis-elements in mediating the CRH transcriptional response In a heterologous TK/CAT promoter/reporter system. c) Fully delineate the transcriptional complex consisting of the -171/-160 element and the protein(s) that bind to it for their role in regulating CRH effects. d) Do mutations in the -234/-133 promoter element which cause major functional changes (aim 2b) have similar effects in the context of the entire homologous POMC promoter? In addition to furthering our understanding of the regulation of this important neuroendocrine gene, these studies will also enhance our knowledge about the molecular and cellular mechanisms by which genes are regulated by intracellular calcium ion, an issue that Is currently poorly understood.

The overall goal of this program is to identify and characterize the cellular and molecular mechanisms which regulate GnRH gene expression. Initial studies will be conducted in an immortalized mouse GnRH neuronal cell model, the GT1-7 cell. We will focus on the PKC and Ca++ signal transduction systems since they have already been shown to elicit significant effects on GnRH transcription, mRNA turnover and secretion. Moreover, Ca++ is a major intracellular path way activated by the excitatory amino acid (EAA) glutamate and we have previously shown that NMDA, has extremely rapid effects on GnRH gene expression at the cytoplasmic level. These findings will be extended to investigations in animals in order to verify that the observed mechanisms are of primary importance in vivo. There are four specific aims: Aim 1:Characterize the elements in the mouse GnRH promoter and the trans- acting factors which are responsible for mediating the negative regulation by the PKC and calcium pathways in the GT1-7 cells. The function of two mouse specific promoter elements will also be identified. Aim 2:Previous studies in cultures of GT1-7 cells have shown that phorbol esters cause a decrease in the stability of GnRH mRNA concomitant with a decrease in poly (A) tail length and a decrease in the number of ribosomes associated with GnRH mRNA. Ca++ ionophores have similar effects on GnRH mRNA stability. Our hypothesis is that GnRH mRNA turnover plays an important role in setting the level of GnRH gene expression. We will determine the mechanism by which activation of the PKC and Ca++ pathways decreases the stability of the GnRH mRNA in GT1-7 cells. Aim 3: Elucidate the mechanism(s) by which rapid changes in GnRH mRNA levels are elicited in the hypothalamus. We will use an EAA paradigm previously shown to significantly modulate GnRH gene expression in vivo and analyze changes in the relative level of polyA and polysome loading of GnRH mRNA in rat hypothalamic neurons. To determine if the post- transcriptional regulatory elements present in GnRH mRNA function in vivo, transgenic mice expressing mutant GnRH mRNA constructs will be created and the effects of EAA treatment analyzed. Aim 4: Using perifusion of GT1 cells, it was reported that different modes of addition of EAAs elicit different responses in GnRH release or in Ca++ activation. We have also seen that secreted GnRH peptide is cleaved to GnRH(1-5) which subsequently antagonizes the NMDA receptor, possibly a mechanism by which GnRH exerts inhibitory ultra-short loop feedback on GnRH neurons. In this study, we will determine if different modes of treatment of GT1-7 cells with EAAs will differentially affect GnRH gene transcription and/or GnRH mRNA stability in a perifusion system.

Neuropeptides are important in transducing changes through their interaction with receptors in the extracellular milieu. Endopeptidase EC 3.4.24.15 (EP24.15) is a soluble metallopeptidase with broad expression in the brain that is involved in neuropeptide metabolism. Enzymes like EP24.15 either: inactivate peptides, convert an inert precursor to an active moiety, or in some instances, convert on bioactive peptide into another with different agonist properties. While the soluble, cytosolic forms of these enzymes have been characterized in purified form, including EP24.15, the properties of the secreted enzyme, much less the secretory mechanisms for the enzymes, have not been extensively studied. Thus, it is important to characterize the enzyme with respect to targeting to the extracellular milieu and the factors modulating enzyme activity extracellularly. This will be accomplished in three Specific Aims. Specific Aim 1: How is EP 24.15 enzyme targeted to the extracellular milieu? Aim 1a. Are there different forms of the EP24.15 derived from the same or different MRNAs? Are the membrane-bound and free ribosome mRNA forms different? What is the structure of the EP24.15 encoding gene? Aim 1b. Determine the secretory pathways targeting the secreted and plasma membrane forms of EP24.15 Which secretory pathway for EP24.15 is subserved by the individual EP24.15 mRNAs? Does mutation of the phosphorylation site and/or cysteinyl mutants change EP24.15 release? Specific Aim 2: Characterize the association between EP24.15 and the plasma membrane or extracellular matrix (ECM). Aim 2a. What is the biochemical differences between plasma membrane and soluble EP24.15? Is the plasma membrane/media form of EP24.15 proteolytically processed? How is EP24.15 associated with the plasma membrane? Aim 2b. Which specific ECM components bind EP24.15 and which EP24.15 are involved in the interaction(s)? Aim 2c. What are the effects of phosphorylation and redox status on EP24.15's association with the extracellular environment? Is the amino terminal domain of EP24.15 important in this association? Specific Aim 3: Determine whether EP24.15 interactions with the cell surface of ECM can modulate EP24.15 activity. Aim 3a. Are the changes in EP24.15 redox-dependent multimerization between different extracellular compartments? Do the mutations which abolish thiol activation of the recomb. enzyme similarly affect the extracellular pool of EP24.15? Aim 3b. Does phosphorylation alter the activity/partition of EP24.15 in the extracellular environment? Determine which form of EP24.15 and the residue on the EP24.15 protein for phosphorylation in vivo.

Parkinson's disease is a progressive neurodegenerative disorder affecting the elderly, characterized by neurodegeneration of nigrostriatal domain neurons. Studies in our laboratory have been focused on identifying factors that are important for the survival and plasticity of dopamine neurons, which could protect them from degeneration or that are important for the survival and plasticity of dopamine neurons, which could protect them from degeneration or enhance compensatory responses. In response to MPTP induced toxicity, midbrain dopaminergic neurons exhibit some regenerative capacity, for young animals treated with the toxin are able to spontaneously recover. However, in the process of normal aging, this regenerative capacity of dopaminergic neurons becomes greatly reduced. We observed in response to MPTP induced degeneration of substantia nigra dopamine neurons in young mice, that there is a robust induction of IL-1 alpha which lasts for nearly two weeks and is associated with collateral sprouting of the ventral tegmental dopamine neurons. In contrast, to middle-aged mice we detected neither an induction of IL-1 alpha or collateral sprouting. To determine whether this induction of IL-1 alpha is necessary for lesion-induced sprouting to occur we are proposing to perform further experiments in mice in which the high affinity IL-1 alpha receptor has been genetically ablated. In further studies we obtained evidence that there may be changes in the extracellular matrix in middle-aged mice that may also contribute to reduced plasticity. Thus, we propose further studies to determine whether ectopic expression of IL-1, by retroviral infection of progenitor cells in middle-aged mice, can stimulate compensatory sprouting of dopamine neurons. In addition, to directly address whether the extracellular environment is play a role in reducing the ability of dopaminergic neurons to collateral sprout, we are proposing to graft fetal dopaminergic neurons into young and middle aged mice and compare the extent of fiber outgrowth. In specific regions of the CNS, neurons die and are being continuously replaced with newly developed neurons. It has recently become appreciated that neural stem cells are more widely dispersed than previously thought and can be induced to proliferate in response to brain injury. We have preliminary data showing that in response to MPTP, newly proliferated cells can be found in the substantia nigra which can have an un-committed phenotype. After longer periods of time after labeling our data suggests the possibility that these cells may eventually differentiate into dopamine neurons. Thus, we propose to establish whether new dopamine neurons are produced in response to injury or can be stimulated to differentiate by ectopically expressing cytokines, which are able to regulate neuronal.

DESCRIPTION (provided by the applicant): We have recently shown in animal models of Parkinson's disease, a progressive neurodegenerative disorder characterized by the degeneration of nigrostriatal dopaminergic neurons, that there is a robust proliferative burst of neuroprogenitor cells in response to the loss of dopamine neurons. However, these cells remain in an undifferentiated state. We have also shown that in response to the degeneration of dopamine neurons in the substantia nigra that the remaining un-injured dopamine neurons are able to compensate for the loss by extending collateral axonal fibers. However, this compensatory response becomes greatly attenuated with increasing age. Therefore, based on these previous results we propose further experiments designed to discover how to induce the differentiation of dopaminergic progenitor cells and injury-induced collateral sprouting in animal models of Parkinson's disease. Integrins are cell surface receptors involved in cell-matrix and cell-cell adhesion interactions in salt organs and play an important role in regulating cell proliferation, survival, and process outgrowth. Studies outside the nervous system indicate that an integration of signals derived from both integrin-ECM interactions and soluble growth factors are required for cellular differentiation. Thus, we hypothesize that ECM molecules expressed in the CNS in combination with growth factors act together to induce neural progenitor cell commitment and differentiation. To test our hypothesis, we propose to characterize which ECM molecules are expressed during the time in development when midbrain dopaminergic progenitors are exiting the cell cycle and making their final commitment to the dopaminergic neuronal fate. In parallel, we plan to culture these progenitor cells on different ECM substrates in combination with fibroblast growth factor-2 (FGF-2) and determine whether the fate of these cells is regulated. We further hypothesize that as the brain develops, the expression of ECM molecules required for the differentiation of neural progenitor cells into dopaminergic neurons becomes down-regulated. To test this idea we plan to culture midbrain progenitor cells on tissue slices containing the substantia nigra from either developing or mature animals. Since our overall goal is to be able to induce dopaminergic progenitor cell differentiation in mature animals, we propose to test whether the induced expression of integrins by retroviral expression vector infection of uncommitted progenitors will induce their differentiation. Finally, we plan to test the hypothesis that age-related differences in ECM molecules play a role in supporting or inhibiting collateral sprouting. Thus, we plan to culture embryonic dopamine neurons on striatal tissue slices collected from control and MPTP mice of different age groups.

leptin; preeclampsia; pathologic process; disease /disorder prevention /control; patient oriented research; human pregnant subject; female; clinical research;