Linda A. Dokas - US grants

Concommitant loss of acetylcholine and somatostatin in the brain during Alzheimer's Disease suggests a functional relationship between this neurotransmitter and peptide. Because acetylcholine and somatostatin are not co-localized to the same cells, receptor-mediated interactions between the two are implied. The cholinergic systems involved are muscarinic and two biochemical processes have been linked to these receptors - increased phosphoinositide turnover and inhibition of adenylate cyclase. Somatostatin analogs are available which can differentiate two types of binding sites on brain membranes, although biochemical correlates of each have not yet been identified. Somatostatin could modulate phosphoinositide metabolism by determining the phosphorylated state of B-50, known to be a regulatory factor in phosphoinositide metabolism. Previous work has suggested that somatostatin acts on B-50 phosphatase and detailed characterization of this enzyme is proposed, since it may be biochemically coupled to a somatostatin receptor. Somatostatin also inhibits adenylate cyclase in several systems. This research will determine whether acetylcholine and somatostatin interact at the level of phosphoinositide/phosphoprotein metabolism or endogenous adenylate cyclase of synaptic membranes. Hippocampal synaptosomes will be prepared, prelabeled with 32 Pi and then incubated with acetylcholine, somatostatin, or both followed by analysis of the 32 P-labeling of phosphoinositide fractions and the protein B-50. Modification of basal and stimulated adenylate cyclase by acetylcholine and somatostatin will be correlated with release of acetylcholine from synaptosomes prelabeled from (3H)- choline. A series of acetylcholine agonists and the selective antagonist pirenzepine will be employed to define the pharmacological specificity of cholinergic responses. Use of analogs known to differentially associate with the two classes of somatostatin receptor in brain membranes will determine which neurochemical systems are linked to each. These studies combine biochemical and pharmacological approaches to define the interaction of acetylcholine and somatostatin with regard to receptor subtypes involved and synaptic compartmentalization. Results so obtained are relevant to the most characteristic neurochemical alterations seen in Alzheimer's Disease. Design of effective therapeutic agents for Alzheimer's Disease will depend on a full definition of the synaptic mechanisms mediated by acetylcholine and somatostatin.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of available data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

During periods of active growth, neurons increase synthesis of a class of proteins that function in the establishment of normal structure and synaptic relations. Among these is GAP(growth-associated)-43 or B-50. A role for this protein in determination of synaptic plasticity is suggested by the co-ordination between its synthesis and axonal growth, its continued expression throughout adult life in the associative areas of the brain and its relationship to long-term potentiation. Neurochemical functions of B-50, which include regulation of phosphoinositide metabolism and Ca2+-dependent processes, are related to its phosphorylation. Well- characterized as a substrate for protein kinase C (PKC), more recent work has demonstrated that B-50 is an in vitro substrate for casein kinase II (CKII) and phosphorylated in intact neurons by an unidentified kinase which recognizes serine or threonine in close proximity to proline residues (MAP kinase-like sites). This project will analyze in vitro phosphorylation of B-50 to identify the latter protein kinase and to examine interactions between the substrate sites in B-50 that allow phosphorylation by each protein kinase. The functional significance of each phosphorylation will be compared to that of PKC with regard to calmodulin binding. The site phosphorylated by each kinase will by characterized with purified substrate and in presynaptic membranes. Dephosphorylation of each substrate site by specific protein phosphatases will be characterized, since such enzymes may limit the extent of in vivo phosphorylation. Preliminary work with growth cones prepared from neonatal rat brains has demonstrated phosphorylation of B-50 on at least one other site in addition to serine 41, the PKC site. These site(s) will be fully characterized. Comparison of B-50 phosphorylation sites and protein kinase activities in growth cones will be compared to those seen in synaptosomes from adult rat brain to determine if the modification of these sites is developmentally-regulated. Analysis of the abilities of PKC, CKII and MAP kinases to phosphorylate B-50 will yield information on how growth stimuli are selectively and temporally coupled to neuronal second messenger systems.