John Oberdick - US grants

Much is known about the molecular and genetic mechanisms controlling the regionalization of the developing brain, but much less is known about how this information ultimately gives rise in maturity to unique brain regions with distinct functions. One strategy to approach this is to dissect the molecular and genetic mechanisms controlling the expression of region-or cell-specific genes whose products contribute directly to a unique functional property of cells in the mature brain. The rodent cerebellum is ideal for the understanding of these mechanisms as there is a rich historical record of natural mutations affecting the development and function of this brain region. In addition much is known about the precise neurobiological functions of its constituent. The purpose of our proposed experiments will be to determine the molecular mechanisms of expression of the L7/Pep-2 gene in the rodent cerebellum. In the mature brain this gene is expressed only in cerebellar Purkinje cells and not in any non-brain tissues. In addition, the gene is expressed in a transient pattern of stripes in the cerebellum during the development and this pattern reflects the well know-known zonal wiring diagram of the cerebellum. Furthermore, the L7-Pcp-2 protein carries a GoLoco-domain and functions as a modulator of GDP binding to large heterotrimeric G-proteins suggesting a role of the protein in modulation of Ca2+channels, a major component of information flow in Purkinje cells. We have combined a novel in vivo expression strategy using transgenic mice with comparative genomics to identify key DNA sequence elements within the promoter (expression control region) of the L7/Pcp-2 gene. The data show very high conservation across species of both the sequence and spacing of seven elements within a short region of this promoter. One of these elements is a binding site for the orphan nuclear receptor, RORa, a likely modulator of Ca2+ metabolism genes in bone that is also known to be expressed in Purkinje cells. The aims will be i) to determine more precisely the role of these elements by expression testing in further transgenic mouse studies, ii) to use FPLC purification and MALDI-MS protein sequencing to identify the key factors that bind to these elements, and III) to test the effect of null mutation of these factors on the expression of L7/Pcp-2. These studies provide the best opportunity to-date to identify a precise molecular complex controlling both cell-specific and G-protein pathway-specific gene expression in the brain.

DESCRIPTION (from applicant's abstract): Neurons receive an enormous variety of inputs that are often segregated in discrete domains on their extracellular surface. This heterogeneity of distribution is reflected in the quantities and kinds of proteins that are targeted to different synapses around the cell. Synaptic heterogeneity is also reflected in the differential distributions of selected mRNAs that are transported into dendrites. It is thought that some mRNAs localized within dendrites play a role in synaptic plasticity. The purpose of the experiments described in this proposal is to provide support for the following general hypothesis: Local electrical signals affect the distribution and utilization of mRNAs in neuronal dendrites via cis-acting mRNA sequences, and this level of control is required to provide a means of adapting the cell to changing patterns and levels of electrical stimulation. This proposal will take advantage of the unique properties of a Purkinje cell-specific mRNA, Pcp-2(L7), and of the cerebellar system to investigate the molecular and cellular mechanisms of dendritic routing of mRNAs. A combined in vivo (transgenic mice) and in vitro (direct injection of labeled mRNAs in cerebellar slices) approach will be used to map RNA routing signals (Aim 1). In addition, dynamic aspects of mRNA trafficking and effects due to electrical activity on mRNA routing, localization and utilization will be examined, and these studies will be facilitated by some novel means of analyzing newly synthesized Pcp-2(L7) mRNA (Aim 2). To test the functional relevance of dendritic mRNA a variant form of mRNA that encodes Pcp-2(L7) protein but is not routed into dendrites will be compared to a normally routed mRNA in behavioral rescue experiments in Pcp-2(L7) null mutant mice (Aim 3). Thus, the molecular analysis in Aim 1 will provide tools to study the functional significance of mRNA routing in Aim 3 as well as an assay (direct mRNA injection) for studying extracellular signals that influence intradendritic localization in Aim 2. Likewise, establishment of inducible expression of L7 mRNA and protein in an L7 null mutant background in Aim 3 will enable an additional tool to be used to study stimulatory effects on newly synthesized mRNA and protein in Aim 2.

A preventive pharmacotherapy for neonatal abstinence syndrome PROJECT SUMMARY/ABSTRACT Neonatal opioid dependence is an enormous and growing medical challenge. The goal of this study is to develop a drug therapy that can prevent opioid dependence in utero in order to suppress or reduce neonatal withdrawal (neonatal abstinence syndrome). The target population affected by this research is pregnant women under clinically controlled pain management or under clinical care for opioid dependence (such as methadone maintenance), and their babies. The proposed therapy must not interfere with management of maternal pain/dependence. To achieve this goal we are testing a peripherally-selective neutral antagonist of the mu-opioid receptor, 6?-naltrexol (6BN). We have shown in mice that the drug crosses the placenta and enters the fetal brain at high levels, but is relatively excluded from the maternal brain by the blood brain barrier (BBB). Thus our therapy takes advantage of the undeveloped fetal BBB. We have also shown that the drug enters the developing mouse brain at high levels until at least postnatal day 14, and that the drug can prevent early postnatal dependence behaviors at high potency when co-administered during morphine administration. The first goal of the proposal is to conduct a detailed pharmacokinetic (PK) analysis of 6BN and methadone (MTD) in pregnant mice (Aim 1A). Compartmental models will be developed to better assess and predict drug exposures. The second goal is to perform initial studies of the pharmacodynamics (PD) of 6BN and MTD in mice to determine a dose range of 6BN that prevents neonatal/juvenile dependence (no BBB), but does not affect maternal/adult pain alleviation (with BBB) (Aim 1B). The final goal is to translate the results in mice to a more appropriate model of the human neonatal developmental state: namely, rhesus monkey. A limited PK analysis will be initiated to determine if 6BN can be preferentially delivered to the fetal monkey brain as in mice, the core of the therapy (Aim 2). The project brings together a diverse team of basic researchers and clinicians with complementary skills, expertise, resources and experience to tackle this substantial societal problem.