James Patrick - US grants

The research projects proposed in this application are concerned with two problems, both of which deal with cholinergic synaptic transmission. The first project uses alpha-bungarotoxin, alpha-mambatoxin and additional toxins which block activation of nicotinic acetylcholine receptors on nerve cells to determine the relationship between the receptor and the molecule which binds alpha-bungaratoxin. This project will be conducted first using clonal cell lines and later central nervous system tissue to study structure, metabolism and regulation of acetylcholine receptors on nerve. The second project uses a murine model of myasthenia gravis to address specific questions about the production of paralysis. Immunological, genetic, and morphological experiments will use our documented strain dependence of paralysis as a means of examining separately the various mechanisms which might cause paralysis. Neuromuscular junctions from high and low responder strains will be studied in vitro to determine effects of antibodies on receptor degradation.

The goal of the work is to understand, at the molecular level, how the interaction of excitable cells results in the formation and muturation of sites of neurotransmission. The approach to this problem is to understand the structure and function of the acetylcholine receptor and the mechanism by which the innervating neuron controls the properties of this neurotransmitter. In the past, the research has focussed upon the acetylcholine receptor at the neuromuscular junction, but recent advances in the molecular biology of the receptor now make it possible to study the nicotinic acetylcholine receptor and cholinergic transmission in the central nervous system. We now have several cDNA clones which encode proteins that we propose are neuronal nicotinic acetylcholine receptor Alpha-subunits. We will determine whether these clones encode a protein which can assemble with or without additional subunits to form a ligand gated ion channel. Initially these experiments will be performed using expression in oocytes or mamallian cell lines in conjection with our available clones encoding the muscle receptor subunits and will provide physiological access to the neuronal nicotinic acetylcholine receptors. We will isolate the neuronal analogues of the muscle Beta-, Gamma-, and Delta-subunits for use in similar expression studies over the long term. Hybrid Alpha-subunits composed of portions of the muscle and neural Alpha-subunits will be constructed to determine which portions of the Alpha-subunits contribure which functions to the receptor oligomer. Antibodies against the several neural Alpha-subunits will be made and used to determine the distribution of nicotinic cholinergic transmission in the central nervous system and, in conjunction with in situ hybridization, will allow us to study the formation and maturation of synapses in the central nervous system.

Receptor proteins that bind nicotine with high affinity and specificity are found in the brain tissue of humans and experimental animals and may be important biochemical mediators of nicotine dependence in tobacco users. The major goals of this proposal are to use monoclonal anti-idiotypic (anti-id) antibodies as molecular probes to study neuronal nicotinic receptor structure and function, and to determine the relationship between cell receptors and active site structures of enzymes involved in nicotine metabolism. The anti-id will be used in immunohistochemical and radioimmunological methods to study the localization and expression of receptors on rat and human brain, and on cultured primary fetal rat cortical cells and PC12 cells, and in immunoaffinity chromatography to isolate receptor by nicotine displacement for gas-phase sequencing and structural analysis. The anti-id complementarity determining regions (CDRs) also will be sequenced and peptides corresponding to the antigen recognition sites synthesized and tested along with the intact anti-id and their F(ab')2 and Fab fragments for their ability to mimic nicotine binding to the receptors and to modulate Ca++ influx into the rat neuronal cells as an expression of nicotine-like functional activity. Computer molecular modeling incorporating id, anti-id, and receptor sequence data and ligand binding specificity, along with similar data for CDR-related peptides will be used to develop a model of the various ligand-acceptor complexes based on energy minimization and molecular dynamics routines. For receptor site mapping, the primary structures and receptor binding activity of the id and anti-id CDR peptides will be compared with binding site sequences derived from immunoaffinity purified or gene-cloned rat and human receptors, and site-directed mutagenesis will be used to make specific amino acid substitutions in the receptor combining site to determine structural requirements for ligand binding. These experimental results will be compared with computer predicted optimal structures for ligand-antibody and ligand-receptor complexes. In similar studies, the ability of anti- nicotine anti-id and anti-id prepared from antibodies to a major metabolite, cotinine, will be used to derive peptide mimics of their respective ligands as hepatic and adrenal cytochrome P-450 inhibitors on the hypothesis that antibody, cell receptor, and enzyme active sites are structurally interrelated through "internal image" anti-id determinants.

Nicotine is a drug of abuse that presumably exerts its effects through its interactions with specific nicotine receptors in the brain. Chronic exposure to nicotine results in an increase in the abundance of nicotine binding sites in the brains of both rats and humans. We have discovered, cloned, and sequenced ten members of the gene family that encodes subunits of the neuronal nicotinic receptors. In this application we propose a means of using these clones to study the metabolism and regulation of the nicotine binding sites in the brain. This approach uses our cDNA clones as templates for the synthesis of the extracellular domains of each receptor subunit in bacteria and then uses these bacterially expressed proteins to generate antibodies in rabbits. Affinity chromatography on columns containing subunit specific peptides results in antibodies able to distinguish between the extracellular domains of each receptor subunit. These antibodies and radioactive peptides are used to create quantitative assays sufficiently sensitive to determine amounts of specific receptor subunits in milligram quantities of brain. We will use these assays to explore the metabolism of the nicotinic receptor and the mechanisms that produce the up-regulation of nicotine binding sites following chronic nicotine use. Our goal is to distinguish between the different mechanisms that might generate more nicotine binding sites. We will also use the antibodies in a more qualitative fashion to determine the location on neurons of nicotinic receptors of specific subunit composition. The long-term goal of this research is to understand how the location of the binding sites that are up-regulated during chronic drug treatment relates to the processes that lead to addiction. These experiments will address issues fundamental to nicotine addiction and to the cellular mechanisms that lead to dependence on the drug.

DESCRIPTION: This is a renewal of a project going into its 19th year, which was last reviewed in 1986. The applicant has isolated clones of several alpha and beta subunits of neuronal nicotinic receptors, sequenced these clones, determined anatomical distribution of the subunits in the CNS, expressed clones in frog oocytes, and described channel functions for most of the subunits when expressed as alpha/beta combinations or as single subunits in the case of the alpha7 subunit. The applicant now proposes to use these nucleic acid probes, and antibodies which he has generated, to examine a series of issues relating to functional receptor composition, assembly, disposition on the cell surface, and role in synapse formation. The first specific aim will involve the development of a transient expression system in mammalian cells which will allow various subunit combinations to be expressed in order to examine issues of subunit pairing, function (particularly as related to calcium permeability) and targeting of subunits to particular cell surfaces. Particular interest is given in the proposal to the alpha7 subunit because of several interesting properties it demonstrates including inhibition by bungarotoxin, the ability to produce in oocytes a functional channel by itself, and the high calcium permeability of the homo-oligomeric channel. Specific Aim 2 will examine the anatomical distribution of alpha7 on cultured neurons, the calcium permeabilities associated with expected areas of alpha7 subunits, and the role of alpha7 activation on neurite outgrowth. Another observation made with the alpha7 subunit is that the ability of the subunit, by itself, to produce a functional channel in oocytes is prevented by exposure of the cells to the peptidyl-prolyl isomerase inhibitor cyclosporin A, thus implicating a role for this enzyme in functional receptor production. In Aim 3 the applicants will test the hypothesis that this enzyme is involved in homo- oligomeric receptors such as the alpha7 receptor. This will be approached through studies examining direct physical association of the enzyme with the subunit, by studies involving mutagenesis of the prolines in alpha7 and other subunits, and through functional studies of receptors formed in oocytes and transfected cells.

Baylor College of Medicine (BCM) has experienced tremendous growth in clinical research activities during the last five years. Although the College has remained steadfast in its commitment to the highest ethical standards in the conduct of human research, it resources have been challenged by the dramatic increase in the variety and volume of clinical investigation and in the heightened public pressures to assure ethical conduct of research. To respond to this explosion of scientific inquiry, the Office of Research at BCM developed a new approach to managing information and documentation of human research subject protections. In August 2001, BCM instituted an electronic protocol management system, the Biomedical Research Assurance Information Network (BRAIN). BRAIN permits electronic submission of protocols for initial and continuing IRB review and facilitates reporting and tracking all key actions for each protocol. The Office of Research Assurance and Compliance Services in cooperation with the IRB Chairs and members has initiated a program for measuring performance of institutional components in maintaining a clinical research environment which fosters; 1) research that minimizes all possible risks to subjects; 2) respectful informed consent; 3) knowledgeable research personnel; and 4) informed and knowledgeable research volunteers. Funding provided through RFA 02-003 has allowed BCM to significantly expand the educational components of BRAIN for clinical research personnel during protocol development, for IRB members during protocol review and for the general public. The financial resources from funding of this application would allow BCM to further expand the types of data that are collected from this electronic platform, to facilitate conflict of interest reporting, to establish data-driven performance improvement standards guided by regulatory, accreditation and best-practice criteria, and to share the educational materials already developed with IRBs in the Houston area. The specific objectives of this initiative are as follows; 1) to develop an electronic conflict of interest reporting, tracking and management system linked to BRAIN that allows more effective communication between the IRB and the Conflict of Interest Committee and that augments current human subjects protections initiatives; 2) to incorporate a common hierarchical dictionary of medical terms into the BRAIN system to provide consistency in the medical and lay language descriptions of clinical research protocols and in adverse event reports to improve process and compliance monitoring and communication among investigators, IRB and the community; 3) to develop and implement a separate service function focused on ongoing assessment of ethical, regulatory and accreditation standards; 4) to provide Assurances and Compliances consultative services and educational materials developed at BCM to Texas Medical Center and Houston area institutional IRBs that currently have either limited or no formal affiliations with Baylor College of Medicine.