Development, maintenance, and regulation of the processes by which nerve cells communicate with one another, termed neurotransmission, depends on the expression of the appropriate neurotransmitter genes. Therefore, elucidation of the mechanisms involved in the regulation of expression of these genes is fundamental to our understanding of neurobiology. Cholinergic neurons, a class of nerve cells that uses as its neurotransmitter a chemical called acetylcholine (ACh), are essential for multiple functions of the nervous system, including the control of skeletal muscles, autonomic activity, the central regulation of movement, and processes underlying learning and memory. These cells express two proteins: choline acetyltransferase (ChAT), responsible for ACh synthesis, and the vesicular ACh transporter (VAChT), responsible for ACh transport into storage vesicles. Remarkably, the genes encoding these proteins are organized into an evolutionarily conserved, integrated unit: the cholinergic gene locus. This unique genomic organization, which likely provides for coordinated regulation of expression of ChAT and VAChT, also conveniently permits the study of the regulation of cholinergic properties with a single, focused approach. The molecular mechanisms underlying the complex regulation of the cholinergic gene locus remain largely unknown. Specifically, it is not known if coordination of VAChT and ChAT expression occurs because the two genes are adjacent to one another but regulated independently, or if the cholinergic gene locus is regulated by one integrated mechanism. The overall goal of the studies described in this proposal is to identify the molecular, biochemical, and cellular mechanisms that regulate the coordinated expression of the cholinergic gene locus. In order to realize this goal the studies will be performed on SN56 cells, which have been characterized extensively in this laboratory and which can be considered the best experimental model of cholinergic neurons currently available. The experimental approach will employ molecular biology techniques. Specifically, the coordinated regulation of ChAT and VAChT expression will be investigated using an artificial gene called a double reporter gene construct in which the sequences encoding VAChT and ChAT have been replaced with two different genes whose expression can be readily detected. The part of the gene locus that controls expression of the genes will remain, and its activity can be measured by monitoring expression of the reporter genes. These constructs will be introduced into the SN56 cells and the activity of the reporter genes as well as of the expression of the endogenous ChAT and VAChT will be determined. Once it is established that regulation of expression of the double reporter gene construct mimics that of ChAT and VAChT, the structure of the construct will be systematically altered in order to determine those segments that provide for the coordinated expression of ChAT and VAChT. The information thus obtained will contribute to current understanding of the cholinergic system, of the mechanisms regulating neurotransmission, and of the fundamental mechanisms involved in regulation of expression of clustered genes.
