Michael J. Bannon - US grants

No Abstract Provided

haloperidol; neurotransmitter biosynthesis; psychotropic drugs; dopamine; Parkinson's disease; genetic transcription; corpus striatum; basal ganglia; drug adverse effect; messenger RNA; gel electrophoresis; radioimmunoassay; high performance liquid chromatography;

DESCRIPTION (provided by applicant): Neurons utilizing dopamine (DA) as a neurotransmitter constitute a rare neurochemical phenotype but nevertheless play an important role in regulating locomotion, motivation, cognition and hormone release. The DA transporter (DAT) is a plasma membrane transport protein that controls the spatio-temporal domains of DA neurotransmission by rapidly reaccumulating DA that has been released into the extracellular space. A wide spectrum of neurological and psychiatric disorders, including drug abuse, Parkinson's disease, schizophrenia, affective disorders, and attention deficit hyperactivity disorder is thought to involve DA systems and the DAT. The DAT is an important target for therapeutic and illicit drugs (e.g. methylphenidate, buproprion, amphetamine, and cocaine), and serves as the point of entry for DA-specific neurotoxins. DAT radioligand binding provides an in vivo measure of DA cell integrity and can be used to monitor the efficacy of therapeutic interventions in neurodegenerative disease. The Aims of this project are to identify the silencing element(s) and cognate transcription factor(s) that repress transcription of the human dopamine transporter gene in non-dopaminergic cells, as well the mechanism by which the transcription factor nurrl activates human dopamine transporter gene transcription in dopamine neurons. It is likely that a greater understanding of the regulation of the DAT expression will impact the diagnosis and treatment of a number of neuropsychiatric disorders.

The dopamine (DA) hypothesis of schizophrenia suggests that at least some of the symptoms of schizophrenia are related to a functional DA over- activity in some forebrain area (s) . Antischizophrenic agents produce both their therapeutic and adverse effects through the blockade of DA receptors. Conversely, the motor and cognitive deficits seen in Parkinson's disease are linked to the loss of forebrain DA and are attenuated by DA replacement therapy. Yet the effects of DA and dopaminergic agents on the functional output of DA-innervated forebrain neurons remains largely uninvestigated. Neurons containing the tachykinin neuropeptides substance P and neurokinin A (substance K) are intimately associated with forebrain DA systems, both as afferents to DA cells and as targets of DA innervation. The proposed research will determine the transcriptional and/or post-transcriptional mechanism(s) underlying the observed DAmediated changes in basal ganglia preprotachykinin (PPT, i.e. tachykinin peptide-encoding) mRNA. Since multiple PPT mRNAs encode different combinations of tachykinins with different biological activities, the effects of altered DA output on the proportion of the various PPT mRNAs, as well as the PPT gene primary transcript levels and the rate of PPT gene transcription, will be studied. Parallel experiments will determine the effects of altered DA transmission on PPT gene expression in limbic brain nuclei receiving dense DA innervations. Acutely dissociated cell preparations will help to establish whether or not the multiple DA receptors which alter PPT mRNAs are located directly on striatal tachykinin neurons. Further experiments using primary neuronal cultures will reveal the signal transduction mechanisms underlying PPT gene regulation and its modulation by DA receptors. Thus the proposed experiments will elucidate the cell and molecular biology of PPT gene expression and its regulation by DA, and will clarify whether alterations in PPT gene expression are potentially involved in the pathologies and therapeutic responses seen in Parkinson's disease and schizophrenia.

DESCRIPTION (from applicant's abstract): The long term objective of this project is to elucidate the molecular and cellular biology dopamine transporter (DAT), a plasma membrane protein thought to control synaptic levels of the neurotransmitter dopamine. Many of the effects of cocaine and other psychostimulants are mediated through interaction with the DAT. Since there are apparently major differences in the cocaine-responsiveness of the DAT in rat and human brain, it is necessary to accurately describe the nature of cocaine-induced changes in the DAT in human brain before this phenomenon can be successfully modeled. The proposed experiments will determine changes in the abundance of DAT mRNA and DAT protein in well- characterized postmortem tissue from cocaine overdose victims using quantitative in situ hybridization histochemical, radio- immunocytochemical and immunobloting techniques. Studies will be then extended to include the serotonin and norepinephrine transporters, physiologically important but understudied cocaine-binding proteins. We have recently sequenced a portion of the human DAT gene and tentatively identified a number of potential regulatory elements. The transcription factor(s) which may underlie the cocaine-responsiveness of the DAT gene will be identified by quantification of transcription factor protein, mRNA and binding activity within human DA cells in cocaine overdose victims and matched control subjects. An understanding of DAT regulation and cocaine-induced changes in human DAT gene expression may shed light on the molecular basis of cocaine addiction, an affliction affecting greater than 1% of the U.S. population. Elucidating the regulation of the DAT may shed light of other pathophysiological states as well, since DAT expression is altered in Parkinson's disease, Alzheimer's disease, normal aging, schizophrenia, Tourette's syndrome, and Lesch-Nyhan disease.

dopamine transporter; genetic promoter element; cocaine; brain stem; neuropharmacology; transcription factor; human genetic material tag; transfection; site directed mutagenesis; laboratory rat; genetic manipulation; tissue /cell culture;

Desensitization to a persistent extracellular signal is a critical characteristic of G protein signaling and multiple mechanism exist to achieve this aim. Regulators of G protein signaling (RGS) proteins are a newly identified class of proteins that play an important role in the desensitization process by acting on G protein subunits. RGS9 is unique among RGS proteins in that it is nearly exclusively expressed in regions of the brain that receive dopaminiergic innervation. Furthermore, RGS9 is uniquely down regulated in response to dopamine release after treatment with amphetamine. Although the exact in vivo function of RGS9 remains unclear, the involvement of dopaminergic neurotransmission in many neuropsychiatric disorders including Parkinson s disease, schizophrenia and drug abuse points to RGS9 as a potential therapeutic target for these disorders. This proposal will provide an integrated analysis of RGS9 expression, regulation and function. We will identify the neuronal cell subtypes that express RGS9 in human and rat basal ganglia. Furthermore, we will examine alterations in RGS9 expression by acute and cronic manipulation of dopaminergic and glutamatergic neurotransmission. RGS9 has recently been cloned and is a complex protein with 3 identifiable domains in addition to its RGS domain. The functions of these various domains are not yet clear but they are likely to be involve din determining the specificity of interactions with other signaling molecules. Molecular reagents, including antibodies, epitope tagged RGS9 expression constructs and domain mutations have been generated that will allow a structure-function analysis of RGS9. These analyses will include the subcellular localization of the RGS9 proteins, its interaction with different G protein subunits and identification of novel binding partners for RGS9. A better understanding of RGS regulation and function will help to clarify how dopamine and dopaminergic agents produce their effects. Our specific aims our: Specific Aim 1. Determine the cellular localization and dopaminergic regulation of RGS9 in the striatum. Specific Aim 2. Identify and analyze RGS signaling partners. Specific Aim 3. Analyze RGS9 targeting and function.