Degeneration or malfunction of brain cholinergic neurons underlies the pathophysiology of a variety of mental disorders: Alzheimer's disease (AD) attacks the most human of all brain functions; cognition, language and social awareness; tardive dyskinesia, a chronic neurological syndrome, is a common side-effect of neuroleptic drugs, which are used to treat mental illness; Korsakoff's dementia develops as a side-effect of alcoholism. Other diseases characterized by cholinergic dysfunction include Motor Neuron Disorders, Huntington's Chorea, and Familial Dysautonomias. Crucial to the development of experimental approaches to study these diseases, and to design treatment strategies, is the establishment of a homogeneous cell preparation which expresses most aspects of the cholinergic phenotype. The goal of the studies described in this application is to characterize the cholinergic properties of a cell line (SN56.B5.G4) developed by hybridization of neuroblastoma cells with mouse brain septal neurons (the neuronal population which is important in memory processing and which degenerates in AD). The activity of choline acetyltransferase, choline uptake, acetylcholine (ACh) storage in vesicles and ACh release in these cells will be studied. The effects of growth factors on the development of the cholinergic phenotype in these cells as well as the effects of pharmacologic agents affecting ACh turnover will be examined. Further, the studies will explore a metabolic peculiarity of cholinergic neurons that might make them especially vulnerable to certain disease processes; their ability of use membrane choline-containing phospholipids as a precursor for ACh. If this process is activated for a prolonged period of time, pathological changes in neuronal membranes might occur, impairing neuronal function or viability. We have previously shown that membrane phosphatidylcholine (PC) is a source of choline for ACh synthesis in human neuroblastoma (LA-N-2) cells and we have obtained preliminary evidence that the choline is liberated from PC by phospholipase D (PLD). Because this enzyme is stimulated by muscarinic receptor activation, we postulate that, in cholinergic synapses, where these receptors are localized, PLD may be activated by ACh, leading to generation of choline which may be taken up by the presynaptic terminals and converted into ACh. We will investigate the regulation of this pathway by a) neuronal firing rate, b) extracellular choline concentration, c) growth factors, d) pharmacological agents that affect ACh synthesis and release, and e) agents that effect phospholipid metabolism. Most experiments will be performed using radiolabeled precursors of ACh and of choline-containing phospholipids and tracing the metabolic fate of the labeled precursors by purifying their metabolites with high performance liquid chromatography. The proposed studies will establish new experimental approaches (which do not involve experimental animals) to study cholinergic functions and will provide new information on the mechanisms regulating ACh synthesis and release.
