Available evidence supports a role for decreased cholinergic function in memory loss and cognitive impairments in the natural aging process and Alzheimer's disease. Our findings show a selective loss of the M2 muscarine receptor subtype on presynaptic cholinergic terminals along with choline acetyltransferase in advanced Alzheimer's disease and retention of the postsynaptic Ml subtype. Cholinergic therapy for the treatment of Alzheimer's dementia must therefore be directed at the M1 receptor mechanism. The overall goal of this application is to define the properties of M1 and M2 receptor subtypes using biochemical and immunological techniques. The proposed work will resolve a discrepancy between pharmacological, anatomical and physiological evidence in favor of two muscarine receptor subtypes, and biochemical evidence for one common receptor protein subunit, by confirming or denying the existence of distinct (whole) receptor proteins. Muscarine receptors from rabbit hippocampus and brainstem, which are rich (approximately 90%) in M1 and M2 subtypes, respectively, will be solubilized in digitonin and purified by a combination of chromatographic techniques. Receptors will be characterized biochemically by size, isoelectric point, amino acid composition, and peptide maps. The role of sulfhydryl and disulfide groups in ligand binding and receptor conformation will be examined. Monoclonal antibodies will be produced to soluble receptor fractions from rabbit hippocampus and brainstem. Antibodies will be screened for their ability to recognize receptors in various forms such as native, denatured, free in solution or immobilized on plastic. Antibodies will be used to confirm or deny the existence of two distinct receptor proteins by the ability of the antibodies to cross-react with the two distinct receptor preparations. Antibodies will be tested for their ability to recognize the receptor proteins in various forms, i.e. as pure proteins, denatured, bound to ligands, or in receptor-effector complexes. Antibodies will also be used for immunoaffinity purification of the receptor proteins which can be further studied biochemically. Finally, antibodies that show specificity of binding to either the M1 or M2 subtypes will be used for immunohistochemical localization of these receptors in tissue sections from rabbit and human brains. These studies will provide useful information on changes in muscarine receptors in normal aging, senile dementia and Alzheimer's disease.
