This project is designed to test the hypothesis that specific areas of the brain such as substantia nigra and basal ganglia are especially vulnerable to oxidative stress and that their response to biochemical oxidative reactions (or oxidative stress) is defective in Parkinsons disease (PD) as well as in normal aging. Mitochondrial and synaptic membranes may lose their function due to inadequate response to oxidation an-or from direct damage induced by cytotoxic products of oxidation (e.g., cholesterol oxidation products). Levels of oxidation products and loss of function could be small under steady state conditions. Therefore, we will perturb the system by inducing oxidation of membranes in vitro (using primarily endogenous oxidants) in order to amplify existing differences. Our preliminary studies show that mitochondrial cholesterol is more oxidizable in PD than in controls. Mitochondrial and synaptosomal fractions will be oxidized by incubation with oxidizing agents (e.g., arachidonic and linoleic acid hydroperoxide plus metal ions like iron, mixtures of iron and ascorbic acid, nitric oxide with and without superoxide etc.). Different levels of oxidation of membranes can be defined by the extent of oxidation of two membrane lipids - tocopherol and cholesterol. Since the profile of cholesterol and tocopherol oxidation products would be characteristic for various types of oxidations, information on the mechanism of oxidation will be obtained. Synaptosomes and mitochondria from young and old rats, and PD and control human autopsy brain samples will be compared in all studies. Similar studies on oxidation of platelets (as an accessible model of neuronal membranes in humans) from PD and control subjects will be conducted. The extent of oxidation of membranes will be monitored and quantitated by determining the concentrations of tocopherol and cholesterol and their oxidation products as well as by estimating thiobarbituric acid reactive substances and the decline in total suIfhydryl concentrations. Oxidizing conditions which yield maximal differences between the control and experimental groups will be established first and then the cholesterol and tocopherol oxidation products will be isolated, identified, and quantitated by GC and GC-MS. A study of the effect of monoamine oxidase(MAO) on mitochondrial oxidation will be done by incubating mitochondria with dopamine, norepinephrine or serotonin (substrates for MAO) which releases in situ hydrogen peroxide and/or other oxidants derived from it. The oxidation will be followed and oxidation products characterized as with brain fraction& The effect of MAO inhibition by deprenyl upon the oxidation will also be studied. Number of similarities exist between the uptake of serotonin by platelets and brain. Serotonin uptake by platelets and synaptosomes at various levels of oxidation will be studied to examine the effect of oxidation upon membrane function. The data will be used to determine whether membrane damage induced by oxidative stress exists in PD and/or in normal aging. The data from the platelet studies may also provide a laboratory tool for the preclinical diagnosis of PD.
