John Merlie, PhD - US grants

The vetebrate muscle nicotinic ACh receptor is very likely the architect of a family of related receptor molecules. The polypeptide subunits are probably encoded by a multigene family. Whereas electroplax ACh receptors or electric fish and embryonic or denervated muscle can be purified in sufficient quantity for detailed biochemical analysis, the putative nACh receptors in the central nervous system and the muscle synapse specific form occur in extremely small quantities and are very difficult to purify. Protein chemical methods cannot identify the relatedness of the non-abundant ACh receptor types or elucidate the molecular bases for the differences among ACh receptors. We propose to isolate by recombinant DNA techniques the cDNA's for each of the embryonic mouse muscle ACh receptor subunits. These cloned cDNA molecules will be used to characterize the nucleic acid and derive protein sequence; to characterize the expression of the ACh receptor mRNA's in different tissues; and to begin to analyze the organization of ACh receptor genes in the genome.

Experiments are proposed which will determine mechanisms governing synthesis and assembly of acetylcholine receptor. The nicotinic ACh-receptor is an integral membrane glycoprotein of muscle which binds Ach released by the nerve. The mechanism by which ACh binding results in a change in membrane permeability culminating in muscle contraction is under intensive study in a number of laboratories. Mechanisms governing receptor synthesis and destruction also deserve intensive study because they are important to the overall function of AChR during development, disease, and regeneration. The synthesis of ACh-receptor as well as other specific proteins is regulated by nerve induced muscle activity. My goal is to study the regulation of synthesis and assembly of the ACh-receptor at the molecular and cellular level. Studies will include the development of immunochemical methods for detection of nascent receptor polypeptides and receptor synthesis in cell free protein synthesizing systems. Combination of these techniques will provide a means for direct study of receptor synthesis at the translational and transcriptional level. Studies of ACh-receptor regulation should serve as a model for the study of other receptors. Control of receptor synthesis may be involved in the regulation of synapse formation, and thereby in the mechanism of establishment of synaptic specificity. Regulation of ACh-receptor content is perturbed in the experimental autoimmune animal model for myasthenia gravis. Studies of this experimental model may lead to improved therapeutic treatment of the human disease. They may also serve in understanding other receptor diseases.

Formation and maintenance of the neuromuscular junction (NMJ) involves the coordinated regulation of gene expression, post-translational processing, assembly of supramolecular protein complexes and protein degradation of a number of specific components. Until recently, the nicotinic acetylcholine receptor (AChR) served as the primary and certainly best studied example of a specific component of the NMJ. Indeed, studies of AChR have led to a firm appreciation of the importance of the delicate balance of synthetic and degradative processes that maintain this synapse. The clinical symptoms of autoimmune, as well as some co genital types of myasthenia gravis, can be understood in terms of their effects on the site-density of AChR at the synapse. In the past several years, monoclonal antibody and recombinant DNA reagents have become available for. the neural cell adhesion molecule (N-CAM), the 43 kDa receptor associated protein (RAPsyn), the junction specific basal lamina component (JS-1) and acetylcholinesterase (AChE); all are interesting and unique components of the NMJ in their own right. Here, we propose to characterize the basic mechanisms involved in the post-translational processing, assembly and stabilization of the AChR, RAPsyn and JS-l molecules. Also, we will study the mechanisms responsible for synapse specific transcriptional regulation of the embryonic (alpha, beta, gamma, and delta) and adult (epsilon) subunits of the AChR as well as RAPsyn, JS-1 and AChE. We will employ DNA mediated gene transfer and transgenic animal techniques for these studies.

This award will support collaborative research in molecular neurobiology between Dr. John P. Merlie, Washington University and Dr. Emmanuel Jover, Unite de Membranes Biologiques et Toxines Animales, French National Center for Scientific Research, Marseille, France. Preliminary experiments employing cDNA cloning indicate the existence of at least four mRNA's for mouse brain Na channels and one for mouse muscle. The investigators propose to extend the screening of existing cDNA libraries to identify yet undiscovered sequences. In addition, mRNA specific probes will be derived from the cDNA clones and used to characterize mRNA distribution in tissues during development and in cell cultures derived from brain and muscle. Finally, they will begin to prepare full length clones for studies of function by expression. The group in Marseille will carry out RNA analysis. At Washington University Dr. Merlie will continue to screen for new cDNA clones, and to sequence the novel clones that are identified. Full length clones for expression will be prepared and expressed in fibroblasts for pharmacological study in Marseille. Dr. Merlie has made significant contributions to the field of sodium channel physiology and pharmacology. Dr. Jovet and his research group have extensive background in mouse development systems. The results of this project are expected to show how recombinant DNA and related techniques can be applied to the study of ion channels.

DESCRIPTION: (Adapted from the applicant's abstract): The goal of this proposal is to understand the molecular components in the formation and maintenance of that neuromuscular synapse. The investigators plan to take advantage of the transgenic mouse technologies to perturb and thereby ascertain the function of three synapse specific genes, the epsilon subunit of the ACh receptor, the 43K/RAPsyn, and the S-laminin. The specific aims are as follows: In Specific Aim 1 is to determine the functional consequence of constitutive expressing the gamma (embryonic) or/and inactivation of the epsilon (adult) subunit of the ACh receptor in adult muscle. This will test the hypotheses concerning the biological role and physiological importance of the switch in expression from gamma to epsilon subunits in developing muscle. To this end, transgenic mouse lines expressing high constitutive levels of the ACh receptor gamma subunit, or transgenic mouse lines with the epsilon subunits gene inactivated, will be generated. Overt phenotypes such as weakness, fatigue and behavioral abnormalities which might be associated with a myasthenic syndrome will be assayed. Functional expression of the gamma-subunit will be determined by analyzing the time course of mepc (miniature end plate current) decay; whether, or not gamma-subunit expression is associated with cytopathic abnormalities of the NMJ (neuromuscular junction) will be determined; the possibility that persistence of gamma-subunit expression delays or abolishes the elimination of polyneuronal innervation will be examined. In Specific Aim 2 is to determine the consequence of disrupting the function of the 43K/RAPsyn gene. 43K/RAPsyn is highly concentrated beneath the synaptic plasma membrane and is thought to interact directly with the ACh receptor to limit mobility. To test this hypothesis, the transgenic mice lines that overexpress mutant 43K/RAPsyn, or transgenic lines with the 43K/RAPsyn gene inactivated, will be generated. These lines will be used to determine whether high density ACh receptor clusters and synaptic localization occur in the absence of RAPsyn; whether the ACh receptor degradation is altered in RAPsyn-less adult muscle; and whether synaptic ultrastructure, including synaptic folds, form normally in the absence of RAPsyn protein. In Specific Aim 3 is to overexpress or to eliminate the expression of S-laminin in developing and regenerating NMJ's in transgenic mice. S-laminin is a synapse specific component of the basal lamina which may be involved in pre-and post-synaptic membrane interaction. In the transgenic lines overproducing S-laminin, the localization or delocalization of the transgene product S-laminin will be determined. If S-laminin is delocalized, whether there is an effect on the precision of re-innervation will be investigated. Finally, the gross morphological developmental defects caused by S-laminin disruption will be characterized.

laboratory mouse

DESCRIPTION (from the abstract): Over several years, Dr. Merlie has successfully collaborated with Dr. Sanes, applying molecular biology techniques to study development of the neuromuscular junction. In the recent past, common efforts of the two laboratories resulted in the generation of homologous recombination mouse mutants lacking s-laminin or 43k/rapsyn. Knock-out constructs for agrin and the AChR e gene have been introduced into ES cells. In this application, Dr. Merlie proposes to use cutting edge molecular technology to study the role of specific genes in neuromuscular junction formation. He lists the following Specific Aims: 1) To generate a skeletal muscle specific knock-out of the s-laminin gene using the Cre/lox P strategy. 2) To study the role of laminin B1 to s-laminin isoform transition in synapse formation by: (1) Rescue of the s-laminin knock out with transgenic constructs encoding s/B1 chimeras; and, (2) Ectopic expression of s-laminin. 3) To generate skeletal muscle and motoneuron specific knock-outs of agrin. 4) To study the role of AChR isoforms in synapse formation by: (1) Converting the g-subunit to an e-subunit and vice versa; and, (2) Rescue of e-gene knock-out with transgenic g/e chimeras to test the roles of extracellular, channel and intracellular domains.