Derangements in synaptic transmission are an important part of the pathology of several neurological and mental diseases including epilepsy, schizophrenia, depression, and perhaps Alzheimer's disease. Despite the medical significance of synaptic transmission and the important roles of synapses in information processing and storage in the brain, relatively little is known about the molecular composition of the key synaptic organelles involved in transmission or about the mechanisms by which the functions of these organelles are regulated. The proposed research involves a study of the molecular structure of synapses in the central nervous system. It focuses on the identification and study of proteins associated with the postsynaptic density (PSD), a large, fibrous specialization of the submembrane cytoskeleton that adheres to the postsynaptic membrane opposite presynaptic terminals. PSDs are especially prominent in central nervous system synapses. There are several hypotheses about PSD function that lead to specific predictions about the nature of PSD-associated proteins. It has been suggested that the PSD may act as an anchor, participating in the adhesion of the presynaptic terminal to the postsynaptic site, or in the clustering of receptors beneath release sites. It may also act as a scaffold upon which regulatory molecules such as protein kinases are positioned to control receptor sensitivity or channel conductances. We have cloned and sequenced one prominent protein component of a subcellular fraction that is highly enriched in PSDs. We have termed the protein PSD-95. PSD-95 is homologous to a Drosophila tumor suppressor protein (dlg) that is associated with intercellular junctions, called septate junctions, that form between developing epithelial cells in insects. Disruption of the dlg gene blocks formation of septate junctions and results in uncontrolled growth of developing epithelial cells. The homology of PSD-95 with dlg supports the idea that postsynaptic density proteins are involved in regulation of synaptic adhesion. It also reveals a previously unsuspected evolutionary relationship between septate junctions and synaptic junctions. We will extend our study of PSD-95 by determining it subcellular location at the electron microscope level. PSD- 95 and the dlg protein contain three distinct structural domains, including a sarc homology 3 domain and a guanylate kinase domain. We will determine the molecular functions of these structural domains, and begin to study the cellular roles of PSD-95 by inhibiting its expression in primary neuronal tissue cultures with the use of antisense oligodeoxynucleotides. We will continue to use microchemical methods to determine the sequences of other prominent proteins associated with the PSD fraction. PSD-associated proteins will be fractionated by detergent extraction, deglycosylation, and gel electrophoresis. Proteins will be transferred to nitrocellulose filters and trypsinized while bound to the paper. Tryptic fragments will be purified by high pressure liquid chromatography and sequenced. The partial sequences will be used to obtain complete sequences by established recombinant DNA methods. We will search sequence data bands to determine whether newly identified proteins are homologous to other proteins of known function. Antibodies specific for each PSD protein will be prepared and used to determine whether the protein is located in the PSD in vivo. The detailed structural information obtained in this study will provide an improved conceptual framework for future studies of synaptic transmission in the central nervous system.
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