Richard L. Rotundo - US grants

The importance of acetylcholinesterase (AChE) lies in its physiological role in neurotransmission, its prominence as a marker for nerve-muscle interactions, and because it is a complex family of oligomeric forms with multiple subcellular locations. Studies of this enzyme will detail how muscle cells regulate the synthesis and assembly of synaptic components in particular, and in broader terms, will provide specific information concerning the mechanisms of regulation and localization of membrane and secreted proteins. The goal of this proposal is to determine the molecular and cellular mechanisms regulating synthesis, assembly, degradation, and localization of the multiple oligomeric forms of AChE in muscle. The overall objective is to determine the relative contributions of the many post-translational events in regulating AChE localization and metabolism. The specific aims are to: 1) determine whether differences in primary structure alone account for the different oligomeric forms and subcellular localizations of AChE; these studies entail isolating isotopically labeled AChE polypeptides from different subcellular compartments and comparing their primary sequences by peptide mapping; 2) determine the structural differences between active and inactive AChE molecules and the cellular mechanisms involved in sorting the two classes; these studies employ a variety of pulse/chase paradigms in conjunction with other biochemical methods to identify modifications, and to determine when and where in the cell these modifications occur; 3) identify the subcellular location of the several AChE forms by indirect immunofluorescence and electron microscopy following differential extractions and enzymatic treatments; 4) study the specific mechanisms of intracellular degradation, assembly, and processing of the AChE polypeptide chains in order to 5) study the role of muscle activity in regulating the synthesis, activation, assembly, transport, and ultimately localization of the multiple AChE forms.

DESCRIPTION (Adapted from the applicant's abstract): Acetylcholinesterase (AChE) is an important marker for understanding the development, organization and regulation of the neuromuscular synapse. Studies of AChE have provided novel and fundamental information towards understanding how nerves regulate skeletal muscle, and studies of the synaptic collagen-tailed AChE form provided knowledge about the formation of the synaptic basal lamina. Dr. Rotundo and colleagues have now developed several new probes and techniques for studying AChE biogenesis, targeting and regulation that will provide new information about the structure and assembly of this prototypical and accessible synapse. Their specific aims are: (1) to continue transcriptional regulation studies to test the hypothesis that the different AChE transcripts arise from different activity-dependent promoters; to study the activity dependent expression of the synapse-specific collagen-like tail subunit by RNase protection, nuclear run-on, and in situ hybridization; (2) to determine the molecular mechanisms responsible for targeting AChE to the synapse including testing the hypothesis that localized exocytosis occurs using secretable Green Fluorescent Protein-AChE chimeric molecules; determining the organization of AChE and nicotinic receptor molecules on the cell surface using new fluorescent and biotinylated ligands that they have synthesized that reveal new relations between the two types of molecules; and testing the hypothesis that complexes of synaptic basal lamina components including AChE attached to perlecan HSP attached to alpha-dystroglycan are organized intracellularly prior to externalization; and (3) continuing their studies on the second messenger systems involved in transducing membrane depolarization into changes in AChE gene regulation, and their studies indicating the presence of muscarinic receptors in skeletal muscle linked to diacylglycerol production and ultimately regulation of AChE expression at the transcriptional and translational levels. These studies will help better understand how nerves control muscle function at the molecular level, and what happens to muscle when it loses the regulatory influence of the nerves.

Neurons synthesize, sort, and transport numerous membrane proteins for localization to highly specialized functional domains on their plasma membranes. The molecular mechanisms whereby they accomplish this targeting are unknown. The long range-goal of this project is to determine the molecular signal(s) responsible for targeting neuronal membrane proteins to their appropriate destinations on the cell surface and the mechanism whereby they are retained within selected regions of the plasma membrane. As a first step in understanding these mechanisms we have chosen to study the processing, transport, and localization of acetylcholinesterase (AChE) in tissue-cultured CNS neurons. AChE is the enzyme that hydrolyses the acetylcholine released by cholinergic nerves. This enzyme exists as a complex family of oligomeric forms, a subset of which are rapidly transported down the axon. In addition, a single integral membrane form of AChE is targeted to the surface plasma membrane in tissue cultured neurons where it is localized in clusters along the neurites. It is likely that the mechanism(s) employed by neurons to sort and transport AChE molecules will be shared by most if not all rapidly transported neuronal membrane proteins, whereas the final targeting will depend upon specific characteristics of subsets of membrane proteins. These are basic studies that will lead to an understanding of how all nerve cells sort and transport membrane proteins to specific regions of their plasma membranes. Our specific aims during the tenure of this proposal are: 1) to determine where, when, and how the signal specifying membrane localization of AChE in CNS neurons occurs; 2) to clone and identify full length cDNAs encoding the AChE polypeptides from chicken CNS neurons using a full length Torpedo AChE cDNA as a probe, 3) to express these cDNAs in mouse L cells to study the fate of the chicken AChE polypeptides, and 4) to inject synthesized AChE, VSV G-glycoprotein, and IL2 receptor mRNA into identified neurons of the leech (Hirudo medicinalis) to study the transport and localization of an identified rapidly transported protein and foreign membrane proteins in vivo.

Neurons synthesize, sort, and transport numerous membrane proteins for localization to highly specialized functional domains on their plasma membranes. The molecular mechanisms whereby they accomplish this targeting are unknown. The long range goal of this project is to determine the molecular signal(s) responsible for targeting neuronal membrane proteins to their appropriate destinations on the cell surface and the mechanism whereby they are retained within selected regions of the plasma membrane. As a first step in understanding these mechanisms we have chosen to study the processing, transport, and localization of acetylocholinesterase (AChE) in tissue-cultured CNS neurons. AChE is the enzyme that hydrolyses the acetylcholine released by cholinergic nerves. This enzyme exists as a complex family of oligomeric forms, a subset of which are rapidly transported down the axon. In addition, a single integral membrane form of AChE is targeted to the surface plasma membrane in tissue cultured neurons where it is localized in clusters along the neurites. It is likely that the mechanism(s) employed by neurons to sort and transport AChE molecules will be shared by most if not all rapidly transported neuronal membrane proteins, whereas the final targeting will depend upon specific characteristics of subsets of membrane proteins. These are basic studies that will lead to an understanding of how all nerve cells sort and transport membrane proteins to specific regions of their plasma membranes. Our specific aims during the tenure of this proposal are: 1) to determine where, when, and how the signal specifying membrane localization of AChE in CNS neurons occurs; 2) to clone and identify full length cDNAs encoding the AChE polypeptides from chicken CNS neurons using a full length Torpedo AChE cDNA as a probe, 3) to express these cDNAs in mouse L cells to study the fate of the chicken AChE polypeptides, and 4) to inject in vitro synthesized AChE, VSV G-glycoprotein, and IL2 receptor mRNA into identified neurons of the leech (Hirudo medicinalis) to study the transport and localization of an identified rapidly transported protein and foreign membrane proteins in vivo.

DESCRIPTION (provided by applicant): Acetylcholinesterase (AChE), the enzyme that terminates cholinergic transmission in the nervous system, is the target of organophosphate nerve agents and pesticides. Inactivation of AChE can result in rapid debilitation or death, thus the use of easily synthesized nerve agents such as soman and sarin by terrorists looms as a potentially catastrophic event. Our laboratory has recently shown that amino terminal peptides derived from the non-catalytic AChE targeting subunits stabilize the newly-synthesized AChE molecules and prevent their degradation in skeletal muscle. This in turn dramatically increases active enzyme and specifically AChE at the neuromuscular synapse when administered to living mice. This increase is rapid and sufficient to protect the animals from exposure to 2x LD50 DFP. Recent preliminary studies in our lab have uncovered small molecules that can enhance AChE folding, and an additional study has provided a novel mechanism for replacing damaged AChE at the neuromuscular synapse. Our specific aims on this project are; 1) to continue our studies on AChE-inducing/stabilizing peptides in vivo using our mouse model to optimize dose and delivery when administered before or after exposure to the surrogate nerve agent DFP; additional studies will test the efficacy of the PRAD-KDEL peptides against soman and tabun; 2) We have identified several ligands that can increase expression of active AChE by enhancing its folding; these compounds will be studied in cultured cells to determine their mechanism of action and test the hypothesis that they act in a manner analogous but not identical to the PRAD-KDEL peptides; possible synergies will be determined; 3) test the hypothesis that these ligands can increase AChE expression in vivo by enhancing folding and stabilization using our mouse model; and 4) Determine the ability of our novel AChE replacement approach using organophosphate resistant enzyme to protect mice from exposure to organophosphate nerve agents and their ability to improve survival when administered after exposure. Together these studies will lead to novel methods for increasing AChE expression in vivo for protection against organophosphate compounds as well as enhancing the rate of recovery following exposure. PUBLIC HEATLTH RELEVANCE: Acetylcholinesterase (AChE) is the enzyme that terminates neuromuscular transmission and is the target of organophosphate nerve gases and some pesticides. Our research is aimed at developing novel therapies to restore active AChE following its inactivation using a combination of peptides and drugs that increase the production of enzyme in the brain and muscles, as well as replacing damaged molecules with new ones.