Paul M. Salvaterra - US grants

We have prepared a panel of monoclonal antibodies specific for the neurotransmitter biosynthetic enzyme choline acetyltransferase. Anti-Drosophila choline acetyltransferase antibodies react with the enzyme active site while antibodies directed against the rat brain enzyme react with a small highly conserved non-active site region of the enzyme. Some of these antibodies cross react with both the Drosophila and rat brain enzymes and possibly with the human enzyme as well. We propose to study the function and significance of the highly conserved immunogenic region of choline acetyltransferase by using specific monoclonal antibodies as probes of the enzyme surface structure. We have also begun a molecular characterization of choline acetyltransferase protein from Drosophila and obtained information on the amino acid sequence of certain tryptic peptides. This limited structural information will be used to design synthetic oligonucleotide "probes" to identify and clone the gene for choline acetyltransferase. Once we have accomplished this goal we will be able to obtain complete structural information about the enzyme and begin to understand the control of its expression in developing, adult and senescent nervous system. Structural information and nucleic acid hybridization probes developed for the Drosophila gene will be used to clone the mammalian gene for choline acetyltransferase. The results of these proposed studies should be useful in understanding normal and abnormal chemical synaptic transmission at the molecular and cellular level in normal and pathological nervous system. Particularly relevant to this latter goal of our studies is the reported involvement of abnormal choline acetyltransferase levels in patients affected with senile dementia of the Alzheimer's type and the availability of structural gene mutants of Drosophila.

Our research program is designed to understand the molecular logic responsible for the correct spatial and temporal expression of choline acetyltransferase (ChAT). We also propose to study the biological significance of ChAT regulation by relating molecular features of ChAT expression to animal behavior. Using genomic clones containing putative regulatory sequences for ChAT, we will continue to map the important cis DNA regulatory elements in P-element transformed Drosophila by observing the expression pattern of a beta-galactosidase reporter gene. We are particularly interested in elements which our initial studies have indicated may be spatially and/or temporally specific for subsets of cholinergic neurons. We will test several potential mechanisms responsible for the differential expression patterns we have seen. Fusion genes of modified ChAT cDNA and ChAT regulatory DNA (and in some cases the reporter gene) will also be incorporated into transgenic animals and analyzed for their ability to produce appropriate levels of ChAT activity as well as correct cellular and subcellular distribution. We also plan to investigate the behavioral consequences of restoring wild type ChAT in animals with a mutant ChAT genetic background. By analyzing behaviors, of both adult and larval stage Drosophila, we will attempt to define specific subsets of cholinergic neurons which play a critical role in mediating a particular behavior. "Rescued cells" will be identified using immunocytochemical and/or in situ hybridization techniques. Our studies will provide basic information which could have relevance to protocols now being developed to genetically engineer cells for neurotransmitter replacement therapies in neurodegenerative diseases such as Alzheimer's.

The overall goal of our proposed work is to understand the genetic and epigenetic strategies as well as the molecular mechanisms used to regulate expression of neurotransmitter specific genes. Appropriate expression of neurotransmitter specific genes determines the phenotypic properties of a neuron as well as its functional state and thus underlies the formation and operation of specific neuronal circuits. Inappropriate expression will result in defective neurotransmission and may contribute to the etiology or pathology of neurotransmitter related diseases. it will be essential to have precise gene regulatory information to properly evaluate the contribution of inappropriate expression to disease states and also to design effective gene therapeutic approaches to treat neurotransmitter related diseases. Our recent work has provided the tools, uncovered some of the strategies and begun to address the mechanisms regulating expression of choline acetyltransferase (ChAT; EC2,3,1,6), the biosynthetic enzyme for acetylcholine production. The ChAT gene (Cha) has recently been discovered to be part of a more complex genetic locus also coding for the vesicular acetylcholine transporter (VAChT) which packages transmitter into synaptic vesicles. The genomic organization of these two distinct but related genetic functions is unique and has been conserved from Drosophila to humans. our proposed studies will attempt to define the significance of this unique genomic organization for the cholinergic locus using Drosophila as a model system. We plan continued identification and analysis of transcriptional control elements and cognate transcription factors regulating expression of the cholinergic locus using P-element "rescue" of mutant phenotypes and biochemical experiments. We will confirm the importance of pdm1/dPOU19 as a key transcriptional regulator of the cholinergic locus, analyze peripheral nervous system regulatory motifs and attempt to define the combination of DNA regulatory elements which are both necessary and sufficient for cholinergic expression. Our preliminary results indicate that cholinergic specificity may be determined by an accessory factor and we thus plan to identify and clone the putative cholinergic co-activator of pdm1/dPOU19. We will also determine the extent and nature of co-ordinate regulation between ChAT and VAChT using immunocytochemistry, in situ hybridization and quantitative methods on wild-type and mutant Drosophila. New aspects of cholinergic locus regulation will be investigated by analyzing suppressor of Cha15 mutations by genetic, transgenic and biochemical methods. We also plan to investigate post-transcriptional regulatory mechanisms for ChAT and VAChT by focusing on translational control mechanisms. Our final goal is to determine the consequences of altered cholinergic gene expression on higher ordered visual behaviors and learning and memory tasks using P-element transformants with abnormal ChAT mini-gene expression patterns.

choline acetyltransferase; membrane transport proteins; gene expression; acetylcholine; synaptic vesicles; suppressor mutations; nucleic acid sequence; complementary DNA; plasmids; visual perception; genetic regulatory element; transcription factor; memory; tissue /cell culture; Drosophilidae; laboratory mouse; genetically modified animals; molecular cloning; immunocytochemistry; in situ hybridization;